Enzyme and Microbial Technology 68 (2015) 15–22
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Fabricating polystyrene fiber-dehydrogenase assemble as a functional biocatalyst Hongjie An, Bo Jin ∗ , Sheng Dai School of Chemical Engineering, The University of Adelaide, 5005 SA, Australia
a r t i c l e
i n f o
Article history: Received 30 June 2014 Received in revised form 20 September 2014 Accepted 25 September 2014 Available online 5 October 2014 Keywords: Biocatalysis Electrospun polystyrene fiber Nitration Enzyme immobilization
a b s t r a c t Immobilization of the enzymes on nano-structured materials is a promising approach to enhance enzyme stabilization, activation and reusability. This study aimed to develop polystyrene fiber-enzyme assembles to catalyze model formaldehyde to methanol dehydrogenation reaction, which is an essential step for bioconversion of CO2 to a renewable bioenergy. We fabricated and modified electrospun polystyrene fibers, which showed high capability to immobilize dehydrogenase for the fiber-enzyme assembles. Results from evaluation of biochemical activities of the fiber-enzyme assemble showed that nitriation with the nitric/sulfuric acid ratio (v/v, 10:1) and silanization treatment delivered desirable enzyme activity and long-term storage stability, showing great promising toward future large-scale applications. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Immobilization of enzymes on nano-structured surfaces has been recognized as a technically promising and cost-effective approach to enhance enzyme stability, activity and reusability [1]. The nanostructured materials offer several intrinsic advantages, such as the larger surface areas that allow a higher loading of enzymes and the enhanced mass and energy transfer efficiency in a bioreactor system. Nano-biocatalysis, in which enzymes are incorporated into nanostructured materials, has emerged as a rapidly growing area [2–4]. Recent development in nanotechnology has provided a wealth of diverse nano-scaled scaffolds that could be used as the support for enzyme immobilization. Those nanostructures, including nano-porous media, nano-fibers [5], nano-tubes and nano-particles, have manifested great efficiency in manipulating the nano-scale environment of the enzymes and thus promise exciting advantages for improving enzyme performances [6]. Nanofibers can create a microenvironment which could enhance the mass transfer of substrate from the reaction medium to the enzyme active sites [2]. Among the nanostructured materials examined for biocatalytical applications, nano-structured polymer fibers (NPF) offer many outstanding characteristics, including high enzyme loading capability and highly homogenous dispersion in liquid phase [7]. In addition, the high porosity and interconnectivity
∗ Corresponding author. Tel.: +61 8 83137056; fax: +61 8 83036222. E-mail address: [email protected] (B. Jin). http://dx.doi.org/10.1016/j.enzmictec.2014.09.010 0141-0229/© 2014 Elsevier Inc. All rights reserved.
endow nano-structured fibers with lower hindrance for mass transfer. The NPF surfaces can be modified to improve enzyme stability and activity. The specific surface characteristics, discrete nanostructures, low costs and ease of fabrication provide exciting opportunities to develop a feasible technology for enzyme-based bioprocesses in the presence of NPF support. Polystyrene (PS) is usually functionalized to make its surface capable of covalent conjugation with proteins. A number of studies have been reported on the PS surface modification by introducing new functional groups, such as hydroxyl, amino, carbonyl, and carboxyl to change its polarity and wettability, so that proteins can be easily grafted to the surface. In practical, those hydrophilic groups are introduced by the surface treatment of ion irradiation [8], plasma [9], electron beam or UV [10,11]. Page et al. modified the surface of PS beads by the nitrating process using sulfuric and nitric acids, followed by the reduction of nitro groups [12]. They confirmed the resulting amine groups were able to immobilize antibodies. Moreover, enzymes have also been reported to better retain their bioactivity as being immobilized via extended spacer arms using the one-step aqueous silanization chemistry to introduce amino groups to microtiter wells for cell growth [13]. Electrospun fiber has attracted an increasing research attention due to its various applications as biocatalyst scaffolds [5]. Electrospun polystyrene fibers (EPSNF) are desirable for enzyme immobilization due to their non-toxicity, low cost and good mechanical properties. To date, the functional groups of EPSNF used for enzyme immobilization are typically introduced by electrospinning the blender of PS and other polymers with functional
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Scheme 1. Consecutive bioconversion of CO2 to methanol catalyzed by three dehydrogenases.
groups such as COOH [14]. However, the dispersion capability of such fibers in aqueous solution is limited, which significantly influence further enzyme loading and enzyme activity. There is almost no information available on the direct modification of pure EPSNFs as the biocatalytical scaffolds. Obert and Dave reported an enzymatically coupled sequential conversion of CO2 to methanol in a series of reactions catalyzed by three different dehydrogenases [15]. The process, as shown in Scheme 1, involves an initial reduction of CO2 to formate catalyzed by formate dehydrogenase (Fate DH), followed by reduction of formate to formaldehyde by formaldehyde dehydrogenase (Fald DH), and finally formaldehyde is reduced to methanol by alcohol dehydrogenase (ADH). However, there are a few crucial technical barriers, including separation and recovery, poor stability/activation and product/intermediate contamination of these enzymes, making this multi-dehydrogenation process technically unreliable and economically unfeasible [16]. The high costs related to these dehydrogenases and cofactor (∼$1500/mole of NADH) restricts their applications in an industrial process [17]. In this study, we investigated the EPSNF nitration in the presence of sulfuric and nitric acids. The influence of the acid mixing ratio on the EPSNF surface properties, enzyme loading efficiency, enzyme activity and stability was systematically examined. The modifications were characterized and the system was evaluated by determining the activity of alcohol dehydrogenate (ADH) in the bioconversion of HCHO to CH3 OH, which is one of three dehydrogenation reactions of bioconversion CO2 into methanol (Scheme 1). 2. Materials and methods 2.1. Electrospinning PS stock solutions (20 and 30%, w/w) were prepared by dissolving PS (MW ∼350,000, Aldrich) in N,N-dimethylformamide (DMF, 98%, Sigma–Aldrich). The solutions were shaked at 50 rpm on an orbital shake incubator at room temperature overnight to ensure PS was fully dissolved. The electrospinning of PS fibers was conducted at room temperature. 0.5 mL of PS stock solution was loaded into a 1 mL syringe equipped with a 22-gauge needle. The syringe was horizontally fixed on a syringe pump (NE-300, New Era Pump Systems), and the solutions were electrospun using high voltage supply (Glassman, PS/EL30P01.5-22). Electrospinning process was conducted with a flow rate of 1 mL/h associated with a variety of operating voltages of 20, and 30 kV. A grounded aluminum foil collector was positioned 15 cm from the tip of the needle. After electrospinning, the fibers were dried at 60 ◦ C in vacuum for 24 h before scanning electron microscope (SEM) examination. 2.2. Surface modification and silanization of electrospun PS fibers The surface modification procedure of PS fibers was illustrated in the flowchart of Fig. 1. Electrospun PS fibers (200 mg) were nitrated with various mixtures of concentrated (63%) nitric acid and (98%) sulfuric acids (v/v, 1:1, 2:1, 3:1, 4:1, 5:1, and 10:1) for 2 h at room temperature under mild shaking (50 rpm), followed by the silanization process as described by Raman Suri and Kaur [18]. The nitro fibers were washed three to five times using Millipore deionized water till the pH of 7, and then treated with 2% (v/v) 3-aminoproplytrimethoxysilane (APTMS, Aldrich) aqueous solution at room temperature for 2 h under mild shaking. The resulting PS fibers were washed with deionized water and dried at 60 ◦ C in vacuum for 2 h.
Fig. 1. Flow chart of the preparation of nano-structured polymer enzyme assembles for this study, which include PS fiber acid nitration, silanization, and enzyme immobilization process.
loading efficiency was determined by Bradford assay [19], where bovine serum albumin (Sigma) was used to prepare calibration curve. Enzymatic activity was evaluated by recording the change in absorption at 340 nm using UV–visible spectrophotometer (LIUV-201 Lambda Scientific). The assay system of ADH contained formaldehyde (5–50 M), -nicotinamide adenine dinucleotide phosphate (50–300 M, Sigma), 0.1 M PBS (pH 7.0). The mixture (3 mL) was put into appropriate cuvettes, immediately mixed by inversion and recorded the absorbance A340 every 30 s, continuously for 5 min. For the fibrous enzymes, the reaction mixture containing fibers were put in a 50 mL centrifuge tube. A340 was measured every 30 s by pipetting 3 mL reaction solutions into a cuvettes, and then were returning into centrifuge tube after measurement. Thus the reduction of formaldehyde to methanol can be calculated by determining the amount of NADH consumption. 2.4. Characterization of PS fibers Specimens of the electrospun PS fibers (with and without surface treatment) were analyzed by SEM (Philips XL 30 FEGSEM) associated with an energy-dispersive X-ray spectroscopy (EDXS), Fourier transform infrared spectroscopy (FTIR, Nicolet 6700) and Raman spectroscopy (PRO-785, PeakSeeker). Fluorescence DyLightTM 649 labeled donkey anti-rabbit IgG antibody (excitation 655 nm, and emission 670 nm) was used as a model protein to verify the immobilization capability of surface activated PS fibers using an Olympus BX51 fluorescence microscope.
2.3. Enzyme immobilization and activity assay After APTMS coating, PS fibers (60 mg) were equilibrated in phosphate buffer silane (PBS, 50 mM, pH 7.0) for 2 h, and then mixed with alcohol dehydrogenase (ADH (A7011), Sigma) solution (0.10 mg/mL) containing N(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC, Sigma) and N-hydroxysuccinimide (NHS, Sigma) (10 g/L in PBS, 50 mM, pH 7.0, molar ratio 1:1) at room temperature gently shaking for 12 h. The resulting ADH-immobilized fibers were washed with PBS until no protein was detected in the supernatant. The enzyme
3. Results and discussion 3.1. Electrospinning optimization It has been reported that the morphologies and diameters of EPSNF can be tailored by varying polymer molecular weight (MW),
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Fig. 2. SEM images of electrospun PS fibers. (A) 20% PS at 20 kV, diameter: 400 nm–5 m, (B) 20% PS 30 kV diameter: 1.26 ± 0.13 m, (C) 30% PS at 20 kV, diameter: 2.28 ± 0.05 m, (D) 30% PS at 30 kV, diameter: 1.66 ± 0.02 m.
solvent, concentration, applied voltage, solution viscosity, conductivity, surface tension, flow rate, inner diameter of capillary tips and processing environment, such as humidity and temperature [20]. Previous study of optimization of EPSNF revealed that fine fibers can be prepared from the PS with a large MW and a low concentration [21]. For the PS with a MW of 1,877,000, fibers with the diameter of 100 nm could be produced using a concentration of 0.8 wt% in DMF [21]. The critical concentration to produce beadfree PS nano-fibers increases with dropping MW, and it can be
20 wt% for the PS with a MW of 100,000 [21]. DMF is typically used for the electrospinning of the PS fibers due to its high conductivity and low viscosity [22,23]. Based on the data in literatures, for the PS with MW: 350,000 g/mol, we chose PS concentrations of 20 and 30 wt% in DMF for electrospinning experiments. As shown in Fig. 2, small amount of beads were visualized on PS fibers in 20 wt% PS solution when a 20 kV voltage was applied. On the other hand, beads or spherical structures were not observed for 30 wt% PS spun at the same applied voltage. In addition, by employing the same
Fig. 3. Morphology of PS fibers before surface treatment (A); and after surface nitration with acid ratios of 1:1 (B), 2:1 (C), and 10:1(D). The nitrated PS fibers treated with other acid ratios (HNO3 : H2 SO4 , 3:1, 4:1, and 5:1) are similar to D.
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Table 1 Beads formation and diameter of PS fiber produced under different electrospinning conditions. Concentration
20%
Voltage
20 kV
30 kV
30% 20 kV
30 kV
Beads Diameter (m)
+ 0.4–5
− 1.2 ± 0.3
− 2.2–3.5
− 1.6 ± 0.2
electrospinning voltage of 30 kV, the PS fibers with larger diameter (2.2 ± 0.4 m–3.5 ± 1.2 m) were produced using the 30 wt% PS solution, in comparison with that of 20 wt% PS solution (fiber diameter 1.2 ± 0.3 m). The results of electrospinning were also summarized in Table 1. To generate desirable uniform bead-free PS fibers for enzyme-polymer assemble, the electrospinning parameters were fixed at the PS concentration of 20 wt% and applied voltage of 30 kV. 3.2. Surface activation of PS fibers
Fig. 4. Optical images of nitrated PS fibers in PBS buffer. Pellet formed at high sulfuric acid component (HNO3 :H2 SO4 , 1:1 and 2:1). Treatment with higher mixing ratios of nitric acid results in hairy PS fiber with good solubility.
Fig. 3 shows the morphologies of EPSNF before and after surface nitration treatment using nitric acid and sulfuric acid at different ratios of HNO3 /H2 SO4 varied from 1:1, 2:1 to 10:1. Significant surface structure changes of PS fibers can be observed after nitration with the acid ratios of 1:1 and 2:1 (Fig. 3B and C). For trial using 1:1 HNO3 /H2 SO4 , PS fibers coagulate together, resulting in forming a firm yellow pellet (Fig. 4). Tubercles are formed on the PS fiber surface which was treated using 2:1 HNO3 /H2 SO4 . Structures of the PS fibers being nitrated with the acid ratios of 3:1, 4:1, 5:1 and 10:1 appear similar. Degradation of the PS fiber can be observed during nitriation process for the acid mixtures of 10:1 (Fig. 3D). It was noted that the solubility of the PS fibers in water was gradually enhanced, if the PS fibers were acidified using HNO3 /H2 SO4 at the moderate mixing ratios (Fig. 4). Suri and Kaur reported that the PS microtiter plates acidification using 47% nitric acid (in sulfuric acid) delivered favorable surface for protein adsorption [18]. The nitration process can be described as the electrophilic aromatic substitution, in which the electrophile is the nitronium ion (NO2 + ) generated from nitric acid through the protonation of nitric acid by sulfuric acid and dehydration (Fig. 5) [24]. In the nitration process, sulfuric acid is used as catalyst and may lead to the formation of sulfonated-polystyrene. The resulting yellow nitrated PS fibers contained higher nitrogen content [25].
O HO
+
N
-
3.3. Energy-dispersive X-ray spectroscopy (EDXS) EDXS can be used for elemental analysis of a small sample associated with the SEM. The EDXS results of PS fibers before and after nitration treatment (HNO3 /H2 SO4 , v/v 3:1) were evaluated and are shown in Fig. 6. The weight contents of each element are analyzed using F element as the reference to remove background noise. Oxygen and nitrogen are not observed on the surface of the as-spun PS fibers before surface treatment, while an obvious oxygen peak can be observed for nitro-PS fibers. In addition, weak N and S peaks are also being observed. The nitrogen content of the nitro-PS fobers varies from 2.56 to 0.57% for nitro-PS fiber samples (HNO3 /H2 SO4 , v/v 1:1 and 10:1), suggesting the nitration is far less than one nitrogroup per styrene unit (9.4% from calculation). The elemental molar ratios of N/C, O/C, and S/C from the EDX study on the PS-fibers being treated by various acid mixtures are given in Fig. 6C.
O
H H OSO3 H
Our SEM results also revealed that the mixing acids containing high contents of sulfuric acid (50 and 33 vol%) were not suitable for enzyme immobilization because these fibers were aggregated together, and thus decreased the surface area.
+
+
O
N
-
H
O
H
O
+
HSO4
-
O +
O
+
N
-
H
H2O
+
O
+
N
O
O
CH2 CH
CH2 CH
CH2 CH
n OCH 3 H Si CH2
n
n
APTMS +
O N
O
OCH3 +
PS fiber
- N O O Nitro PS fiber
NH2 3
NO2 APTMS PS fiber
Fig. 5. Schematics of nitration process. The electrophile, the nitronium ion (NO2 + ), is generated from nitric acid through the protonation of nitric acid by sulfuric acid and loss of water. Electrophile substitution takes place when PS fiber attacks the nitronium ions [25].
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Fig. 7. ATR-FTIR spectra of untreated (PS) and nitrated PS fiber surface with varying nitric: sulfuric acids (v/v, 1:1, 2:1, 3:1, and 10:1).
Fig. 6. (A) EDX spectra of PS fibers without modification, (B) Typical EDX spectra of PS fibers after nitration. (C) Element molar ratios of modified PS fibers.
Elemental analysis reveals that the above acid treatment can introduce nitrogen and oxygen to the surface of the PS fibers. Maximal nitro-group introduction is obtained for the treatment with 1:1 nitric/sulfuric acids. The nitrogen contents decrease with the increment of nitric acid ratios in the acid mixture, which indicates the decrease in the NO2 substitution. The strongest oxygen signal was found at a mixing ratio of 3:1, associated with a strong sulfur signal. The experimental results indicate that sulfur was introduced during nitration. The presence of the sulfur indicates the sulfonation reaction on fiber surface. 3.4. Spectroscopic analysis of the nitrated PS fiber IR and Raman spectroscopy were used to identify the functional groups on the surface of the nitrated PS fibers using nitric/sulfuric acid at different ratios. The characteristic polystyrene bands can be found at 1602, 1493, 1451, 1180, 905, 754 and 700 cm−1 in the
IR spectrum of the untreated PS fibers [26]. The nitrated PS fibers showed three new peaks at 1520, 1344 and 855 cm−1 , which were different from that of the untreated PS fibers (Fig. 7). These results agree with previous reports PS beads nitration [12]. The peaks at 1520 and 1344 cm−1 are consistent with the asymmetrical and symmetrical stretches of the NO bonds of nitro group. The peak at 855 cm−1 is associated with the para-substituted aromatics. For the PS fibers treated by nitric/sulfuric acid ratios of 1:1, 2:1 and 3:1, the PS peaks at 1600, 1492, and 1451 cm−1 disappeared, which suggests the partial destruction of PS fibers. Raman spectrum of untreated PS fiber (Fig. 8) shows the characteristic PS bands at the Raman shifts of 620, 1000, 1030, 1070, 1151, 1181, 1201, 1450 and 1601 cm−1 [27]. The nitrated PS fibers reveal different Raman spectra at the peaks of 858, 1110, 1345 and 1598 cm−1 . Those Raman bands agree well with our FTIR results and others [28]. 858 cm−1 is assigned to CN stretching, 1108 cm−1 is the in-plane CH deformation, 1345 cm−1 for NO2 symmetric stretching and 1597 cm−1 for the NO2 anti-symmetric stretching [28]. The small band at 1710 cm−1 might be assigned to the CO stretch of the carbonyl groups. Similar as the surface modification of carbon nanotubes, acidification using high concentration of HNO3 or H2 SO4 leads to partial oxidization.
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Fig. 9. Protein loading efficiency on nitrated PS fibers and activity retention of immobilized enzymes at different acid ratio.
Fig. 8. Raman spectra of untreated (PS) and nitrated PS fiber surface with varying nitric: sulfuric acids (v/v, 1:1, 2:1, 3:1, and 10:1). 785 nm excitation, 1 cm−1 resolution.
In summary, the acid treatment process has a dominant influence on nitro group substitution. Besides the nitro groups, both carboxyl and -SO3 groups can be introduced during the acidification, which gives rise to the improvement in the hydrophilicity of the PS fibers. The as-spun PS fibers are hydrophobic and float on the surface of water, but the nitrated hairy PS fibers can be well dispersed in water as being treated using the mix acids at the ratios of 4:1, 5:1 and 10:1 (Fig. 4). Our results indicate that the surface modification using a mixture of nitric acid and sulfuric acid at a certain ratio not only make the surface more active, but also alter the fiber’s surface wettability or hydrophilicity.
3.5. Enzyme loading Silanization of the EPSNF using APTMS was carried out followed the nitration process. Functional NH2 group can be introduced through the silanization. Proteins are allowed to attach to the amine groups via the EDC/NHS. As shown in Fig. 9, the protein loading efficiency is strongly sensitive to the nitration process of the PS fibers. A high acid ratio of HNO3 /H2 SO4 for the PS nitration resulted in an enhanced enzyme loading efficiency. A loading of 18 mg ADH proteins per g PS fiber can be achieved for the PS fibers treated using 10:1 HNO3 /H2 SO4 . The enzyme loading gradually decreased as sulfuric acid concentration increased. The loaded proteins decreased to 1 mg proteins per g fiber when the HNO3 /H2 SO4 ratio was 1:1. For comparison, protein loading was also conducted on the as-spun PS fibers and nitrated PS fibers without APTMS treatment. Due to the hydrophobic properties of the as-spun PS fibers, proteins could be hardly adsorbed on the surface with the value very close to null. The ADH immobilization on nitro PS fibers showed a lower loading efficiency (1.1 ± 0.3 mg enzyme per gram fiber) although they can be well dispersed in PBS.
As shown in Fig. 9, there is a trend: the higher nitric acid ratio, the higher protein loading efficiency. One may suspect that 100% nitric acid will achieve the highest protein loading efficiency. Theoretically, the sulfuric acid is used as catalyst. The nitration reactions will go very slowly without sulfuric acids. The nitro fiber without silanization gave a low protein loading efficiency. The reason could be that those nitro-groups are less favorable for protein conjugation since the EDC chemistry is highly selective. The lower ADH loading may be also caused by physical adsorption. The physical structures of PS fibers affect the enzyme loading efficiency. SEM images show that the PS fibers were aggregated after nitration using high contents of sulfuric acid. As a result, it was difficult to disperse them in the PBS (Fig. 4). The aggregation of the PS fibers leads to a significant reduction in the surface area, which lowers the following silanization, conjugation and the mass transfer of proteins, thus resulting in the low loading efficiency. The successful conjugation of proteins to the modified PS fibers was further confirmed by fluorescence microscopy and fluorometer (Shimadzu RF-5301PC Spectrofluorometer). Fluorescence labeled DyLightTM 649 donkey anti-rabbit IgG antibody was used as a model protein. Fig. 10A and B shows the fluorescence and optical images of modified PS fibers. No fluorescence can be found for the PS fibers using an excitation wavelength of 649 nm. Fig. 10C and D shows the fluorescence and optical images of fluorescence labeled protein immobilized on the surface of functionalized PS fibers. Spectrofluorometer data also confirmed the protein attachments (Fig. 11) 3.6. Enzyme activity and storage stability of immobilized ADH The activity of free and immobilized ADH are showed in Table 2. The specific activity of the ADH immobilized directly on the nitro PS fibers is about half of the free ADH. The activity retention of enzymes immobilized on the nanofibers was calculated as their specificity activity divided by that of free enzymes. The nitro PS fiber has the loading efficiency of 1.1 mg/g fiber and 51% of activity retention. Enzymes loaded on the APTMS treated PS fibers present a higher loading efficiency (18 mg/g fiber) and a higher activity retention (68.6%), which indicates that the silane linker is favorable for bioconversion. The activity retention under different acids’ ratio was depicted in Fig. 9. PS fibers treated by nitric/sulfuric acid ratios of 10:1, 5:1 and 4:1 exhibited higher activity retention rate (66–69%). The treatment by nitric/sulfuric acid ratios of 1:1, 2:1 and 3:1 showed relative lower activity retention. As shown in Fig. 4, lower nitric/sulfuric acid ratio (HNO3 /H2 SO4 , 1:1–3:1) treatment results in compact structures, and thus the fibers are poorly dispersed in aqueous solutions. These physical morphology changes will end up with poor enzyme loading and activity, and also limit the diffusion of substrates from the reaction medium to the enzymes. In comparison, higher nitric/sulfuric acids
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Fig. 10. Typical fluorescence image of fluorescence labeled proteins (DyLightTM 649 donkey anti-rabbit IgG) on as electrospun PS fibers (A and B) and modified PS fibers (C and D). (A) and (C), fluorescence image excitation at 499 nm. (B) and (D), optical image under white light. Magnification: 1000× (Olympus BX51 fluorescence microscope).
Table 2 Loading efficiency and activity for immobilized ADH on the electrospun PS fibers. Details
Adsorbed enzyme (mg/g)
Specific activity (U/mg)
Activity retention (%)
Free ADH ADH immobilized on nitro PS fiber (10:1) ADH immobilized on APTMS-nitro PS fibers (10:1)
– 1.1 ± 0.3 18 ± 2.1
305.3 155.6 209.4
100 51.3 ± 1.2 68.6 ± 0.7
Fluorescence intensity
ratio (HNO3 /H2 SO4 , 4:1–10:1) treatment provides fibers with good dispersity in aqueous solutions, which are favorable for enzyme loading and can minimize substrate diffusional limitation. Compared to the previous works on enzyme immobilization on electrospun fiber as references cited in the review paper [5], where described the activity retention rate ranging 6–81%, our value of 68.6% activity retention rate is significantly high. Jia et al. used polystyrene fibers functionalized by 4-(dimethylamina)-pyridine as a carrier for ␣-chymotrypsin, and obtained 14 mg/g fiber loading efficiency and 65% enzyme activity retention [29]. Nair et al. used
670
680
690
700
710
720
730
740
750
760
Wavelength (nm) Fig. 11. Fluorescence spectrum of 649 donkey anti-rabbit IgG antibody immobilized PS fibers.
alcohol treated polystyrene copolymer fibers for lipase research, and got high protein loading efficiency (42.4 ± 18.5 mg/g fiber) but only 16.5% enzyme activity retention [30]. The APTMS modification can improve the biocompatibility of system and create a hydrophilic microenvironment which is favorable for enzyme access and conjugation, and thus enhance the specific activity. When the enzyme is immobilized on the nitro PS fiber surface, its dispensability and the capability to access the substrate is limited. However, the remaining activity is still desirable. The reusability and storage stability of the enzymes immobilized on modified PS fibers (HNO3 /H2 SO4 , 10:1) in PBS buffer solution were evaluated. Fig. 12 presents the normalized enzyme activities during 4 weeks storage in fridge. Enzyme activity decreased approximately 42% after the first week storage, but remained at about 33% after 4 weeks storage. During the re-use experiments, the enzyme activity significantly dropped in the first week. We believe that some enzymes immobilized on smaller fragmental fibers might be washed off due to the changes of reaction solutions, and thus resulted in a decrease of total enzyme activity. The loss of immobilized enzymes could be overcome if the fiber-enzyme catalysts are used in a bioreactor sealed by filters. In this case, the immobilized enzymes retained 33% of their initial activities after four repeated batch reactions during storage at 4 ◦ C for 4 weeks without any stabilizer. Although this study showed that the storage and operational stability of the immobilized enzymes are acceptable, the loss of 67% activities
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Fig. 12. Normalized enzyme activities immobilized on nitrated PS fiber after storage in fridge and re-used in several cycles.
cannot be neglected. Immobilized enzymes can be contaminated and degraded by bacteria, protease and peptidases when the biocatalysis are carried out in a non-sterile environment. Further study on the stabilization to provide a microenvironment for the modified enzymes preventing them from denaturation and inactivation needs to be conducted. Due to the ease in enzyme recycling, continuous operation and product purification, the current results encourage the design of nitration modified PS fiber scaffolds toward bioprocess strategy for large-scale applications. 4. Conclusions The modified EPSNF scaffolds toward biocatalysis were evaluated by the model reaction of formaldehyde reduction to methanol catalyzed by ADH. Nitration with highly concentrated nitric acid is desirable for the PS fiber modification and enzyme loading efficiency. The results indicate silanization has a favorable impact on enzyme activity and improves significantly the activity retention of immobilized ADH. The current work suggests the immobilized enzymes demonstrate a promising biochemical activity after several cycles use and long-term storage. Improvement of stability of immobilized enzyme needs further research. This protocol is promising to deliver a cost-effective sustainable fiber-enzyme assemble as a biocatalysis platform. Acknowledgment This study was supported by an exploring research funding from Bionanotechnology Laboratory: Water, Energy and Materials at The University of Adelaide, Australia. References [1] Moehlenbrock MJ, Minteer SD. Introduction to the field of enzyme immobilization and stabilization. In: Minteer SD, editor. Enzyme stabilization and immobilization: methods and protocols. New York: Human Press; 2010. p. 1–7.
[2] Kim J, Grate JW, Wang P. Nanostructures for enzyme stabilization. Chem Eng Sci 2006;61:1017–26. [3] Wang P. Nanoscale biocatalyst systems. Curr Opin Biotechnol 2006;17:574–9. [4] Fang Y, Huang XJ, Peng-Cheng C, Xu ZK. Polymer materials for enzyme immobilization and their application in bioreactors. BMB Rep 2011;44:87–95. [5] Wang ZG, Wan LS, Liu ZM, Huang XJ, Xu ZK. Enzyme immobilization on electrospun polymer fibers: an overview. J Mol Catal B 2009;56:189–95. [6] Kim J, Grate JW, Wang P. Nanobiocatalysis and its potential applications. Trends Biotechnol 2008;26:639–46. [7] Song J, Kahveci D, Chen M, Guo Z, Xie E, Xu X, et al. Enhanced catalytic activity of lipase encapsulated in PCL fibers. Langmuir 2012;28:6157–62. [8] Kim KH, Cho JS, Choi DJ, Koh SK. Hydrophilic group formation and cell culturing on polystyrene Petri-dish modified by ion-assisted reaction. Nucl Instr Meth Phys Res B 2001;175–177:542–7. [9] Ohl A, Schroder K. Plasma-induced chemical micropatterning for cell culturing applications: a brief review. Surf Coat Technol 1999;116–119:820–30. [10] Onyiriuka EC. Electron beam surface modification of polystyrene used for cell cultures. J Adhes Sci Technol 1994;8:1–9. [11] Lu HW, Lu QH, Chen WT, Xu HJ, Yin J. Cell culturing on nanogrooved polystyrene petri dish induced by ultraviolet laser irradiation. Mater Lett 2004;58: 29–32. [12] Page JD, Derango R, Huang AE. Chemical modification of polystyrene’s surface and its effect on immobilized antibodies. Colloids Surf A 1998;132:193–201. [13] Kaur J, Singh KV, Raje M, Varshney GC. Raman Suri C. Strategies for direct attachment of hapten to a polystyrene support for applications in enzyme-linked immunosorbent assay (ELISA). Anal Chim Acta 2004;506:133–5. [14] Nair S, Kim J, Crawford B, Kim SH. Improving biocatalytic activity of enzymeloaded fibers by dispersing entangled fiber structure. Biomacromolecules 2007;8:1266–70. [15] Obert R, Dave BC. Enzymatic conversion of carbon dioxide to methanol: enhanced methanol production in silica sol–gel matrices. J Amer Chem Soc 1999;121:12192–3. [16] Liu W, Zhang S, Wang P. Nanoparticle-supported multi-enzyme biocatalysis with in situ cofactor regeneration. J Biotechnol 2009;139:102–7. [17] Liu W, Wang P. Cofactor regeneration for sustainable enzymatic biosynthesis. Biotechnol Adv 2007;25:369–84. [18] Raman Suri C, Kaur J. Direct hapten coated ELISA for immunosensing of low molecular weight analytes. Protocol Exch 2007;395:126–32. [19] 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. [20] Nitanan T, Opanasopit P, Akkaramongkolporn P, Rojanarata T, Ngawhirunpat T, Supaphol P. Effects of processing parameters on morphology of electrospun polystyrene fibers. Korean J Chem Eng 2012;29:173–81. [21] Eda G, Shivkumar S. Bead-to-fiber transition in electrospun polystyrene. J Appl Polym Sci 2007;106:475–87. [22] Kim K, Kang M, Chin IJ, Jin HJ. Unique surface morphology of electrospun polystyrene fibers from a N,N-dimethylformamide solution. Macromol Res 2005;13:533–7. [23] Jarusuwannapoom T, Hongrojjanawiwat W, Jitjaicham S, Wannatong L, Nithitanakul M, Pattamaprom C, et al. Effect of solvents on electro-spinnability of polystyrene solutions and morphological appearance of resulting electrospun polystyrene fibers. Eur Polym J 2005;41:409–21. [24] McMurry J. Organic chemistry. 6th ed. Belmount: Thomson Learning; 2004. [25] Zenftman H. The nitration of polystyrene. J Chem Soc 1950;38:982–6. [26] Liang CY, Krimm S. Infrared spectra of high polymers. VI. Polystyrene. J Polym Sci 1958;27:241–54. [27] Sears WM, Hunt JL, Stevens JR. Raman spectra at low temperatures and depolarization ratios for styrene and polystyrene. J Chem Phys 1982;77:1639–44. [28] Sacristan J, Reinecke H, Mijangos C, Spells S, Yarwod J. Surface modification of polystyrene films. Depth profiling and mapping by Raman microscopy. Macromol Chem Phys 2002;203:678–85. [29] Jia H, Zhu G, Vugrinovich B, Kataphinan W, Reneker D, Wang P. Enzyme-carring polymeric nanofibers prepared via electrospinning for use as unique biocatalysts. Biotechnol Prog 2002;18:1027–32. [30] Nair S, Kim J, Crawford B, Kim S. Improving biocatalytic activity of enzyme-loaded nanofibers by dispersing entangled nanofiber structure. Biomacromolecules 2007;8:1266–70.
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Fabricating polystyrene fiber-dehydrogenase assemble as a functional biocatalyst Hongjie An, Bo Jin ∗ , Sheng Dai School of Chemical Engineering, The University of Adelaide, 5005 SA, Australia
a r t i c l e
i n f o
Article history: Received 30 June 2014 Received in revised form 20 September 2014 Accepted 25 September 2014 Available online 5 October 2014 Keywords: Biocatalysis Electrospun polystyrene fiber Nitration Enzyme immobilization
a b s t r a c t Immobilization of the enzymes on nano-structured materials is a promising approach to enhance enzyme stabilization, activation and reusability. This study aimed to develop polystyrene fiber-enzyme assembles to catalyze model formaldehyde to methanol dehydrogenation reaction, which is an essential step for bioconversion of CO2 to a renewable bioenergy. We fabricated and modified electrospun polystyrene fibers, which showed high capability to immobilize dehydrogenase for the fiber-enzyme assembles. Results from evaluation of biochemical activities of the fiber-enzyme assemble showed that nitriation with the nitric/sulfuric acid ratio (v/v, 10:1) and silanization treatment delivered desirable enzyme activity and long-term storage stability, showing great promising toward future large-scale applications. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Immobilization of enzymes on nano-structured surfaces has been recognized as a technically promising and cost-effective approach to enhance enzyme stability, activity and reusability [1]. The nanostructured materials offer several intrinsic advantages, such as the larger surface areas that allow a higher loading of enzymes and the enhanced mass and energy transfer efficiency in a bioreactor system. Nano-biocatalysis, in which enzymes are incorporated into nanostructured materials, has emerged as a rapidly growing area [2–4]. Recent development in nanotechnology has provided a wealth of diverse nano-scaled scaffolds that could be used as the support for enzyme immobilization. Those nanostructures, including nano-porous media, nano-fibers [5], nano-tubes and nano-particles, have manifested great efficiency in manipulating the nano-scale environment of the enzymes and thus promise exciting advantages for improving enzyme performances [6]. Nanofibers can create a microenvironment which could enhance the mass transfer of substrate from the reaction medium to the enzyme active sites [2]. Among the nanostructured materials examined for biocatalytical applications, nano-structured polymer fibers (NPF) offer many outstanding characteristics, including high enzyme loading capability and highly homogenous dispersion in liquid phase [7]. In addition, the high porosity and interconnectivity
∗ Corresponding author. Tel.: +61 8 83137056; fax: +61 8 83036222. E-mail address: [email protected] (B. Jin). http://dx.doi.org/10.1016/j.enzmictec.2014.09.010 0141-0229/© 2014 Elsevier Inc. All rights reserved.
endow nano-structured fibers with lower hindrance for mass transfer. The NPF surfaces can be modified to improve enzyme stability and activity. The specific surface characteristics, discrete nanostructures, low costs and ease of fabrication provide exciting opportunities to develop a feasible technology for enzyme-based bioprocesses in the presence of NPF support. Polystyrene (PS) is usually functionalized to make its surface capable of covalent conjugation with proteins. A number of studies have been reported on the PS surface modification by introducing new functional groups, such as hydroxyl, amino, carbonyl, and carboxyl to change its polarity and wettability, so that proteins can be easily grafted to the surface. In practical, those hydrophilic groups are introduced by the surface treatment of ion irradiation [8], plasma [9], electron beam or UV [10,11]. Page et al. modified the surface of PS beads by the nitrating process using sulfuric and nitric acids, followed by the reduction of nitro groups [12]. They confirmed the resulting amine groups were able to immobilize antibodies. Moreover, enzymes have also been reported to better retain their bioactivity as being immobilized via extended spacer arms using the one-step aqueous silanization chemistry to introduce amino groups to microtiter wells for cell growth [13]. Electrospun fiber has attracted an increasing research attention due to its various applications as biocatalyst scaffolds [5]. Electrospun polystyrene fibers (EPSNF) are desirable for enzyme immobilization due to their non-toxicity, low cost and good mechanical properties. To date, the functional groups of EPSNF used for enzyme immobilization are typically introduced by electrospinning the blender of PS and other polymers with functional
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Scheme 1. Consecutive bioconversion of CO2 to methanol catalyzed by three dehydrogenases.
groups such as COOH [14]. However, the dispersion capability of such fibers in aqueous solution is limited, which significantly influence further enzyme loading and enzyme activity. There is almost no information available on the direct modification of pure EPSNFs as the biocatalytical scaffolds. Obert and Dave reported an enzymatically coupled sequential conversion of CO2 to methanol in a series of reactions catalyzed by three different dehydrogenases [15]. The process, as shown in Scheme 1, involves an initial reduction of CO2 to formate catalyzed by formate dehydrogenase (Fate DH), followed by reduction of formate to formaldehyde by formaldehyde dehydrogenase (Fald DH), and finally formaldehyde is reduced to methanol by alcohol dehydrogenase (ADH). However, there are a few crucial technical barriers, including separation and recovery, poor stability/activation and product/intermediate contamination of these enzymes, making this multi-dehydrogenation process technically unreliable and economically unfeasible [16]. The high costs related to these dehydrogenases and cofactor (∼$1500/mole of NADH) restricts their applications in an industrial process [17]. In this study, we investigated the EPSNF nitration in the presence of sulfuric and nitric acids. The influence of the acid mixing ratio on the EPSNF surface properties, enzyme loading efficiency, enzyme activity and stability was systematically examined. The modifications were characterized and the system was evaluated by determining the activity of alcohol dehydrogenate (ADH) in the bioconversion of HCHO to CH3 OH, which is one of three dehydrogenation reactions of bioconversion CO2 into methanol (Scheme 1). 2. Materials and methods 2.1. Electrospinning PS stock solutions (20 and 30%, w/w) were prepared by dissolving PS (MW ∼350,000, Aldrich) in N,N-dimethylformamide (DMF, 98%, Sigma–Aldrich). The solutions were shaked at 50 rpm on an orbital shake incubator at room temperature overnight to ensure PS was fully dissolved. The electrospinning of PS fibers was conducted at room temperature. 0.5 mL of PS stock solution was loaded into a 1 mL syringe equipped with a 22-gauge needle. The syringe was horizontally fixed on a syringe pump (NE-300, New Era Pump Systems), and the solutions were electrospun using high voltage supply (Glassman, PS/EL30P01.5-22). Electrospinning process was conducted with a flow rate of 1 mL/h associated with a variety of operating voltages of 20, and 30 kV. A grounded aluminum foil collector was positioned 15 cm from the tip of the needle. After electrospinning, the fibers were dried at 60 ◦ C in vacuum for 24 h before scanning electron microscope (SEM) examination. 2.2. Surface modification and silanization of electrospun PS fibers The surface modification procedure of PS fibers was illustrated in the flowchart of Fig. 1. Electrospun PS fibers (200 mg) were nitrated with various mixtures of concentrated (63%) nitric acid and (98%) sulfuric acids (v/v, 1:1, 2:1, 3:1, 4:1, 5:1, and 10:1) for 2 h at room temperature under mild shaking (50 rpm), followed by the silanization process as described by Raman Suri and Kaur [18]. The nitro fibers were washed three to five times using Millipore deionized water till the pH of 7, and then treated with 2% (v/v) 3-aminoproplytrimethoxysilane (APTMS, Aldrich) aqueous solution at room temperature for 2 h under mild shaking. The resulting PS fibers were washed with deionized water and dried at 60 ◦ C in vacuum for 2 h.
Fig. 1. Flow chart of the preparation of nano-structured polymer enzyme assembles for this study, which include PS fiber acid nitration, silanization, and enzyme immobilization process.
loading efficiency was determined by Bradford assay [19], where bovine serum albumin (Sigma) was used to prepare calibration curve. Enzymatic activity was evaluated by recording the change in absorption at 340 nm using UV–visible spectrophotometer (LIUV-201 Lambda Scientific). The assay system of ADH contained formaldehyde (5–50 M), -nicotinamide adenine dinucleotide phosphate (50–300 M, Sigma), 0.1 M PBS (pH 7.0). The mixture (3 mL) was put into appropriate cuvettes, immediately mixed by inversion and recorded the absorbance A340 every 30 s, continuously for 5 min. For the fibrous enzymes, the reaction mixture containing fibers were put in a 50 mL centrifuge tube. A340 was measured every 30 s by pipetting 3 mL reaction solutions into a cuvettes, and then were returning into centrifuge tube after measurement. Thus the reduction of formaldehyde to methanol can be calculated by determining the amount of NADH consumption. 2.4. Characterization of PS fibers Specimens of the electrospun PS fibers (with and without surface treatment) were analyzed by SEM (Philips XL 30 FEGSEM) associated with an energy-dispersive X-ray spectroscopy (EDXS), Fourier transform infrared spectroscopy (FTIR, Nicolet 6700) and Raman spectroscopy (PRO-785, PeakSeeker). Fluorescence DyLightTM 649 labeled donkey anti-rabbit IgG antibody (excitation 655 nm, and emission 670 nm) was used as a model protein to verify the immobilization capability of surface activated PS fibers using an Olympus BX51 fluorescence microscope.
2.3. Enzyme immobilization and activity assay After APTMS coating, PS fibers (60 mg) were equilibrated in phosphate buffer silane (PBS, 50 mM, pH 7.0) for 2 h, and then mixed with alcohol dehydrogenase (ADH (A7011), Sigma) solution (0.10 mg/mL) containing N(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride (EDC, Sigma) and N-hydroxysuccinimide (NHS, Sigma) (10 g/L in PBS, 50 mM, pH 7.0, molar ratio 1:1) at room temperature gently shaking for 12 h. The resulting ADH-immobilized fibers were washed with PBS until no protein was detected in the supernatant. The enzyme
3. Results and discussion 3.1. Electrospinning optimization It has been reported that the morphologies and diameters of EPSNF can be tailored by varying polymer molecular weight (MW),
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Fig. 2. SEM images of electrospun PS fibers. (A) 20% PS at 20 kV, diameter: 400 nm–5 m, (B) 20% PS 30 kV diameter: 1.26 ± 0.13 m, (C) 30% PS at 20 kV, diameter: 2.28 ± 0.05 m, (D) 30% PS at 30 kV, diameter: 1.66 ± 0.02 m.
solvent, concentration, applied voltage, solution viscosity, conductivity, surface tension, flow rate, inner diameter of capillary tips and processing environment, such as humidity and temperature [20]. Previous study of optimization of EPSNF revealed that fine fibers can be prepared from the PS with a large MW and a low concentration [21]. For the PS with a MW of 1,877,000, fibers with the diameter of 100 nm could be produced using a concentration of 0.8 wt% in DMF [21]. The critical concentration to produce beadfree PS nano-fibers increases with dropping MW, and it can be
20 wt% for the PS with a MW of 100,000 [21]. DMF is typically used for the electrospinning of the PS fibers due to its high conductivity and low viscosity [22,23]. Based on the data in literatures, for the PS with MW: 350,000 g/mol, we chose PS concentrations of 20 and 30 wt% in DMF for electrospinning experiments. As shown in Fig. 2, small amount of beads were visualized on PS fibers in 20 wt% PS solution when a 20 kV voltage was applied. On the other hand, beads or spherical structures were not observed for 30 wt% PS spun at the same applied voltage. In addition, by employing the same
Fig. 3. Morphology of PS fibers before surface treatment (A); and after surface nitration with acid ratios of 1:1 (B), 2:1 (C), and 10:1(D). The nitrated PS fibers treated with other acid ratios (HNO3 : H2 SO4 , 3:1, 4:1, and 5:1) are similar to D.
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Table 1 Beads formation and diameter of PS fiber produced under different electrospinning conditions. Concentration
20%
Voltage
20 kV
30 kV
30% 20 kV
30 kV
Beads Diameter (m)
+ 0.4–5
− 1.2 ± 0.3
− 2.2–3.5
− 1.6 ± 0.2
electrospinning voltage of 30 kV, the PS fibers with larger diameter (2.2 ± 0.4 m–3.5 ± 1.2 m) were produced using the 30 wt% PS solution, in comparison with that of 20 wt% PS solution (fiber diameter 1.2 ± 0.3 m). The results of electrospinning were also summarized in Table 1. To generate desirable uniform bead-free PS fibers for enzyme-polymer assemble, the electrospinning parameters were fixed at the PS concentration of 20 wt% and applied voltage of 30 kV. 3.2. Surface activation of PS fibers
Fig. 4. Optical images of nitrated PS fibers in PBS buffer. Pellet formed at high sulfuric acid component (HNO3 :H2 SO4 , 1:1 and 2:1). Treatment with higher mixing ratios of nitric acid results in hairy PS fiber with good solubility.
Fig. 3 shows the morphologies of EPSNF before and after surface nitration treatment using nitric acid and sulfuric acid at different ratios of HNO3 /H2 SO4 varied from 1:1, 2:1 to 10:1. Significant surface structure changes of PS fibers can be observed after nitration with the acid ratios of 1:1 and 2:1 (Fig. 3B and C). For trial using 1:1 HNO3 /H2 SO4 , PS fibers coagulate together, resulting in forming a firm yellow pellet (Fig. 4). Tubercles are formed on the PS fiber surface which was treated using 2:1 HNO3 /H2 SO4 . Structures of the PS fibers being nitrated with the acid ratios of 3:1, 4:1, 5:1 and 10:1 appear similar. Degradation of the PS fiber can be observed during nitriation process for the acid mixtures of 10:1 (Fig. 3D). It was noted that the solubility of the PS fibers in water was gradually enhanced, if the PS fibers were acidified using HNO3 /H2 SO4 at the moderate mixing ratios (Fig. 4). Suri and Kaur reported that the PS microtiter plates acidification using 47% nitric acid (in sulfuric acid) delivered favorable surface for protein adsorption [18]. The nitration process can be described as the electrophilic aromatic substitution, in which the electrophile is the nitronium ion (NO2 + ) generated from nitric acid through the protonation of nitric acid by sulfuric acid and dehydration (Fig. 5) [24]. In the nitration process, sulfuric acid is used as catalyst and may lead to the formation of sulfonated-polystyrene. The resulting yellow nitrated PS fibers contained higher nitrogen content [25].
O HO
+
N
-
3.3. Energy-dispersive X-ray spectroscopy (EDXS) EDXS can be used for elemental analysis of a small sample associated with the SEM. The EDXS results of PS fibers before and after nitration treatment (HNO3 /H2 SO4 , v/v 3:1) were evaluated and are shown in Fig. 6. The weight contents of each element are analyzed using F element as the reference to remove background noise. Oxygen and nitrogen are not observed on the surface of the as-spun PS fibers before surface treatment, while an obvious oxygen peak can be observed for nitro-PS fibers. In addition, weak N and S peaks are also being observed. The nitrogen content of the nitro-PS fobers varies from 2.56 to 0.57% for nitro-PS fiber samples (HNO3 /H2 SO4 , v/v 1:1 and 10:1), suggesting the nitration is far less than one nitrogroup per styrene unit (9.4% from calculation). The elemental molar ratios of N/C, O/C, and S/C from the EDX study on the PS-fibers being treated by various acid mixtures are given in Fig. 6C.
O
H H OSO3 H
Our SEM results also revealed that the mixing acids containing high contents of sulfuric acid (50 and 33 vol%) were not suitable for enzyme immobilization because these fibers were aggregated together, and thus decreased the surface area.
+
+
O
N
-
H
O
H
O
+
HSO4
-
O +
O
+
N
-
H
H2O
+
O
+
N
O
O
CH2 CH
CH2 CH
CH2 CH
n OCH 3 H Si CH2
n
n
APTMS +
O N
O
OCH3 +
PS fiber
- N O O Nitro PS fiber
NH2 3
NO2 APTMS PS fiber
Fig. 5. Schematics of nitration process. The electrophile, the nitronium ion (NO2 + ), is generated from nitric acid through the protonation of nitric acid by sulfuric acid and loss of water. Electrophile substitution takes place when PS fiber attacks the nitronium ions [25].
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Fig. 7. ATR-FTIR spectra of untreated (PS) and nitrated PS fiber surface with varying nitric: sulfuric acids (v/v, 1:1, 2:1, 3:1, and 10:1).
Fig. 6. (A) EDX spectra of PS fibers without modification, (B) Typical EDX spectra of PS fibers after nitration. (C) Element molar ratios of modified PS fibers.
Elemental analysis reveals that the above acid treatment can introduce nitrogen and oxygen to the surface of the PS fibers. Maximal nitro-group introduction is obtained for the treatment with 1:1 nitric/sulfuric acids. The nitrogen contents decrease with the increment of nitric acid ratios in the acid mixture, which indicates the decrease in the NO2 substitution. The strongest oxygen signal was found at a mixing ratio of 3:1, associated with a strong sulfur signal. The experimental results indicate that sulfur was introduced during nitration. The presence of the sulfur indicates the sulfonation reaction on fiber surface. 3.4. Spectroscopic analysis of the nitrated PS fiber IR and Raman spectroscopy were used to identify the functional groups on the surface of the nitrated PS fibers using nitric/sulfuric acid at different ratios. The characteristic polystyrene bands can be found at 1602, 1493, 1451, 1180, 905, 754 and 700 cm−1 in the
IR spectrum of the untreated PS fibers [26]. The nitrated PS fibers showed three new peaks at 1520, 1344 and 855 cm−1 , which were different from that of the untreated PS fibers (Fig. 7). These results agree with previous reports PS beads nitration [12]. The peaks at 1520 and 1344 cm−1 are consistent with the asymmetrical and symmetrical stretches of the NO bonds of nitro group. The peak at 855 cm−1 is associated with the para-substituted aromatics. For the PS fibers treated by nitric/sulfuric acid ratios of 1:1, 2:1 and 3:1, the PS peaks at 1600, 1492, and 1451 cm−1 disappeared, which suggests the partial destruction of PS fibers. Raman spectrum of untreated PS fiber (Fig. 8) shows the characteristic PS bands at the Raman shifts of 620, 1000, 1030, 1070, 1151, 1181, 1201, 1450 and 1601 cm−1 [27]. The nitrated PS fibers reveal different Raman spectra at the peaks of 858, 1110, 1345 and 1598 cm−1 . Those Raman bands agree well with our FTIR results and others [28]. 858 cm−1 is assigned to CN stretching, 1108 cm−1 is the in-plane CH deformation, 1345 cm−1 for NO2 symmetric stretching and 1597 cm−1 for the NO2 anti-symmetric stretching [28]. The small band at 1710 cm−1 might be assigned to the CO stretch of the carbonyl groups. Similar as the surface modification of carbon nanotubes, acidification using high concentration of HNO3 or H2 SO4 leads to partial oxidization.
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Fig. 9. Protein loading efficiency on nitrated PS fibers and activity retention of immobilized enzymes at different acid ratio.
Fig. 8. Raman spectra of untreated (PS) and nitrated PS fiber surface with varying nitric: sulfuric acids (v/v, 1:1, 2:1, 3:1, and 10:1). 785 nm excitation, 1 cm−1 resolution.
In summary, the acid treatment process has a dominant influence on nitro group substitution. Besides the nitro groups, both carboxyl and -SO3 groups can be introduced during the acidification, which gives rise to the improvement in the hydrophilicity of the PS fibers. The as-spun PS fibers are hydrophobic and float on the surface of water, but the nitrated hairy PS fibers can be well dispersed in water as being treated using the mix acids at the ratios of 4:1, 5:1 and 10:1 (Fig. 4). Our results indicate that the surface modification using a mixture of nitric acid and sulfuric acid at a certain ratio not only make the surface more active, but also alter the fiber’s surface wettability or hydrophilicity.
3.5. Enzyme loading Silanization of the EPSNF using APTMS was carried out followed the nitration process. Functional NH2 group can be introduced through the silanization. Proteins are allowed to attach to the amine groups via the EDC/NHS. As shown in Fig. 9, the protein loading efficiency is strongly sensitive to the nitration process of the PS fibers. A high acid ratio of HNO3 /H2 SO4 for the PS nitration resulted in an enhanced enzyme loading efficiency. A loading of 18 mg ADH proteins per g PS fiber can be achieved for the PS fibers treated using 10:1 HNO3 /H2 SO4 . The enzyme loading gradually decreased as sulfuric acid concentration increased. The loaded proteins decreased to 1 mg proteins per g fiber when the HNO3 /H2 SO4 ratio was 1:1. For comparison, protein loading was also conducted on the as-spun PS fibers and nitrated PS fibers without APTMS treatment. Due to the hydrophobic properties of the as-spun PS fibers, proteins could be hardly adsorbed on the surface with the value very close to null. The ADH immobilization on nitro PS fibers showed a lower loading efficiency (1.1 ± 0.3 mg enzyme per gram fiber) although they can be well dispersed in PBS.
As shown in Fig. 9, there is a trend: the higher nitric acid ratio, the higher protein loading efficiency. One may suspect that 100% nitric acid will achieve the highest protein loading efficiency. Theoretically, the sulfuric acid is used as catalyst. The nitration reactions will go very slowly without sulfuric acids. The nitro fiber without silanization gave a low protein loading efficiency. The reason could be that those nitro-groups are less favorable for protein conjugation since the EDC chemistry is highly selective. The lower ADH loading may be also caused by physical adsorption. The physical structures of PS fibers affect the enzyme loading efficiency. SEM images show that the PS fibers were aggregated after nitration using high contents of sulfuric acid. As a result, it was difficult to disperse them in the PBS (Fig. 4). The aggregation of the PS fibers leads to a significant reduction in the surface area, which lowers the following silanization, conjugation and the mass transfer of proteins, thus resulting in the low loading efficiency. The successful conjugation of proteins to the modified PS fibers was further confirmed by fluorescence microscopy and fluorometer (Shimadzu RF-5301PC Spectrofluorometer). Fluorescence labeled DyLightTM 649 donkey anti-rabbit IgG antibody was used as a model protein. Fig. 10A and B shows the fluorescence and optical images of modified PS fibers. No fluorescence can be found for the PS fibers using an excitation wavelength of 649 nm. Fig. 10C and D shows the fluorescence and optical images of fluorescence labeled protein immobilized on the surface of functionalized PS fibers. Spectrofluorometer data also confirmed the protein attachments (Fig. 11) 3.6. Enzyme activity and storage stability of immobilized ADH The activity of free and immobilized ADH are showed in Table 2. The specific activity of the ADH immobilized directly on the nitro PS fibers is about half of the free ADH. The activity retention of enzymes immobilized on the nanofibers was calculated as their specificity activity divided by that of free enzymes. The nitro PS fiber has the loading efficiency of 1.1 mg/g fiber and 51% of activity retention. Enzymes loaded on the APTMS treated PS fibers present a higher loading efficiency (18 mg/g fiber) and a higher activity retention (68.6%), which indicates that the silane linker is favorable for bioconversion. The activity retention under different acids’ ratio was depicted in Fig. 9. PS fibers treated by nitric/sulfuric acid ratios of 10:1, 5:1 and 4:1 exhibited higher activity retention rate (66–69%). The treatment by nitric/sulfuric acid ratios of 1:1, 2:1 and 3:1 showed relative lower activity retention. As shown in Fig. 4, lower nitric/sulfuric acid ratio (HNO3 /H2 SO4 , 1:1–3:1) treatment results in compact structures, and thus the fibers are poorly dispersed in aqueous solutions. These physical morphology changes will end up with poor enzyme loading and activity, and also limit the diffusion of substrates from the reaction medium to the enzymes. In comparison, higher nitric/sulfuric acids
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Fig. 10. Typical fluorescence image of fluorescence labeled proteins (DyLightTM 649 donkey anti-rabbit IgG) on as electrospun PS fibers (A and B) and modified PS fibers (C and D). (A) and (C), fluorescence image excitation at 499 nm. (B) and (D), optical image under white light. Magnification: 1000× (Olympus BX51 fluorescence microscope).
Table 2 Loading efficiency and activity for immobilized ADH on the electrospun PS fibers. Details
Adsorbed enzyme (mg/g)
Specific activity (U/mg)
Activity retention (%)
Free ADH ADH immobilized on nitro PS fiber (10:1) ADH immobilized on APTMS-nitro PS fibers (10:1)
– 1.1 ± 0.3 18 ± 2.1
305.3 155.6 209.4
100 51.3 ± 1.2 68.6 ± 0.7
Fluorescence intensity
ratio (HNO3 /H2 SO4 , 4:1–10:1) treatment provides fibers with good dispersity in aqueous solutions, which are favorable for enzyme loading and can minimize substrate diffusional limitation. Compared to the previous works on enzyme immobilization on electrospun fiber as references cited in the review paper [5], where described the activity retention rate ranging 6–81%, our value of 68.6% activity retention rate is significantly high. Jia et al. used polystyrene fibers functionalized by 4-(dimethylamina)-pyridine as a carrier for ␣-chymotrypsin, and obtained 14 mg/g fiber loading efficiency and 65% enzyme activity retention [29]. Nair et al. used
670
680
690
700
710
720
730
740
750
760
Wavelength (nm) Fig. 11. Fluorescence spectrum of 649 donkey anti-rabbit IgG antibody immobilized PS fibers.
alcohol treated polystyrene copolymer fibers for lipase research, and got high protein loading efficiency (42.4 ± 18.5 mg/g fiber) but only 16.5% enzyme activity retention [30]. The APTMS modification can improve the biocompatibility of system and create a hydrophilic microenvironment which is favorable for enzyme access and conjugation, and thus enhance the specific activity. When the enzyme is immobilized on the nitro PS fiber surface, its dispensability and the capability to access the substrate is limited. However, the remaining activity is still desirable. The reusability and storage stability of the enzymes immobilized on modified PS fibers (HNO3 /H2 SO4 , 10:1) in PBS buffer solution were evaluated. Fig. 12 presents the normalized enzyme activities during 4 weeks storage in fridge. Enzyme activity decreased approximately 42% after the first week storage, but remained at about 33% after 4 weeks storage. During the re-use experiments, the enzyme activity significantly dropped in the first week. We believe that some enzymes immobilized on smaller fragmental fibers might be washed off due to the changes of reaction solutions, and thus resulted in a decrease of total enzyme activity. The loss of immobilized enzymes could be overcome if the fiber-enzyme catalysts are used in a bioreactor sealed by filters. In this case, the immobilized enzymes retained 33% of their initial activities after four repeated batch reactions during storage at 4 ◦ C for 4 weeks without any stabilizer. Although this study showed that the storage and operational stability of the immobilized enzymes are acceptable, the loss of 67% activities
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Fig. 12. Normalized enzyme activities immobilized on nitrated PS fiber after storage in fridge and re-used in several cycles.
cannot be neglected. Immobilized enzymes can be contaminated and degraded by bacteria, protease and peptidases when the biocatalysis are carried out in a non-sterile environment. Further study on the stabilization to provide a microenvironment for the modified enzymes preventing them from denaturation and inactivation needs to be conducted. Due to the ease in enzyme recycling, continuous operation and product purification, the current results encourage the design of nitration modified PS fiber scaffolds toward bioprocess strategy for large-scale applications. 4. Conclusions The modified EPSNF scaffolds toward biocatalysis were evaluated by the model reaction of formaldehyde reduction to methanol catalyzed by ADH. Nitration with highly concentrated nitric acid is desirable for the PS fiber modification and enzyme loading efficiency. The results indicate silanization has a favorable impact on enzyme activity and improves significantly the activity retention of immobilized ADH. The current work suggests the immobilized enzymes demonstrate a promising biochemical activity after several cycles use and long-term storage. Improvement of stability of immobilized enzyme needs further research. This protocol is promising to deliver a cost-effective sustainable fiber-enzyme assemble as a biocatalysis platform. Acknowledgment This study was supported by an exploring research funding from Bionanotechnology Laboratory: Water, Energy and Materials at The University of Adelaide, Australia. References [1] Moehlenbrock MJ, Minteer SD. Introduction to the field of enzyme immobilization and stabilization. In: Minteer SD, editor. Enzyme stabilization and immobilization: methods and protocols. New York: Human Press; 2010. p. 1–7.
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