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Encapsulation and immobilization of papain in electrospun nanofibrous membranes of PVA cross-linked with glutaraldehyde vapor
Materials Science and Engineering C 52 (2015) 306–314
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Encapsulation and immobilization of papain in electrospun nanofibrous membranes of PVA cross-linked with glutaraldehyde vapor Iván E. Moreno-Cortez a,b,c, Jorge Romero-García a,⁎, Virgilio González-González b,c, Domingo I. García-Gutierrez b,c, Marco A. Garza-Navarro b,c, Rodolfo Cruz-Silva d a
Centro de Investigación en Química Aplicada (CIQA), Blvd. Enrique Reyna # 140, San José de los Cerritos, Saltillo, Coahuila 25100, México Universidad Autónoma de Nuevo León (UANL), Fac. de Ingeniería Mecánica y Eléctrica (FIME), Av. Universidad S/N, Cd. Universitaria, San Nicolás de los Garza, Nuevo León C.P. 66450, México Universidad Autónoma de Nuevo León (UANL), Centro de Innovación, Investigación y Desarrollo en Ingeniería y Tecnología (CIIDIT), Apodaca, Nuevo León, México d Research Center for Exotic Nanocarbons, Shinshu University, 4-17-1 Wakasato, Nagano 380-8553, Japan b c
a r t i c l e
i n f o
Article history: Received 12 January 2015 Received in revised form 12 March 2015 Accepted 23 March 2015 Available online 25 March 2015 Keywords: Nanofibers Papain Enzyme immobilization Electrospinning
a b s t r a c t In this paper, papain enzyme (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in poly(vinyl alcohol) (PVA) nanofibers prepared by electrospinning. The morphology of the electrospun nanofibers was characterized by scanning electron microscopy (SEM) and the diameter distribution was in the range of 80 to 170 nm. The presence of the enzyme within the PVA nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The maximum catalytic activity was reached when the enzyme loading was 13%. The immobilization of papain in the nanofiber membrane was achieved by chemical crosslinking with a glutaraldehyde vapor treatment (GAvt). The catalytic activity of the immobilized papain was 88% with respect to the free enzyme. The crosslinking time by GAvt to immobilize the enzyme onto the nanofiber mat was 24 h, and the enzyme retained its catalytic activity after six cycles. The crosslinked samples maintained 40% of their initial activity after being stored for 14 days. PVA electrospun nanofibers are excellent matrices for the immobilization of enzymes due to their high surface area and their nanoporous structure. © 2015 Published by Elsevier B.V.
1. Introduction The immobilization of enzymes has evolved into a common technique to extend the catalytic activity of these important biological molecules that usually lose their three-dimensional molecular structure when they are in solution. Papain is a thiol protease commonly found in many organisms, such as plants and fungi. The kinetics and the structure of papain have been well-studied; it is considered the enzyme par excellence to compare the efficiency of various immobilization methods [1]. The applications of papain in many industrial, medical, and pharmaceutical technologies have fostered research to develop a variety of immobilization protocols [2]. This protease has been extensively used in traditional food processing applications as a chill-proofing agent: for instance, during beer-finishing operations in the brewing process and also to tenderize meat in the meat industry [3,4]. Papain has been used as a catalyst to oligomerize hydrophobic amino acids [5–11]. The potential uses of papain include the development of assays and biosensors. For instance, the strong interaction between bioconjugated carbon nanotubes and papain opens up the possibility of biosensors using these materials for the detection of 9-carboxy tetrahydrocannabinol in urine ⁎ Corresponding author. E-mail address: [email protected] (J. Romero-García).
http://dx.doi.org/10.1016/j.msec.2015.03.049 0928-4931/© 2015 Published by Elsevier B.V.
to test marijuana consumption [12]. Kim et al. created a modulated fluorescent sensor, in which Hg+ induced the agglutination of a poly(paraphenyleneethynylene)–papain complex [13]. Recently, several nanomaterials have been used as a matrix for papain immobilization. Among them, we can find magnetic nanoparticles [14], carbon nanotubes [15] and polymeric micro- and nanoparticles [2,16]. A different reported method demonstrated that cross-linked enzyme aggregates (CLEAs) represent a simple and useful enzyme immobilization technique, which was recently applied in the immobilization of papain [17]. Another technique, known as electrospinning, has been used in the immobilization of several enzymes [22] but not for the immobilization of papain. A great deal of information regarding electrospun polymer fibers and their applications can be found in the literature [18]. The process of nanofiber formation by electrospinning can be successfully controlled to obtain long and uniform nanofibers, which can be deposited to form non-woven mats and membranes with open porosity, high surface area and good mechanical properties. Several research groups around the world have used electrospinning in the immobilization of biologically active molecules and cells of commercial and scientific relevance, such as viruses, bacteria and various types of cells that have successfully been immobilized on polymeric electrospun membranes [19,20]. The encapsulated biomolecules have
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been used in important and interesting applications, such as biosensors, drug release systems and tissue engineering [20–22]. The enzymes have a fundamental role in the structure and metabolism of the human body in order to keep cells with optimal performance. Moreover, enzymes are important biomolecules in scientific research, as well as in industrial processes. A variety of enzymes has been encapsulated and immobilized using the electrospinning technique. A few examples are: glucose oxidase, lipase, laccase, lysozyme, lactate dehydrogenase, horseradish peroxidase, fructose dehydrogenase, urease, tyrosinase and cellulase [22–30]. In most of the reported cases of enzyme immobilization using electrospinning, it has been demonstrated that neither the electric field nor the polymer–enzyme interactions present in the electrospinning process affect the catalytic function of the immobilized enzymes. In general, the catalytic function of the immobilized enzymes presents an improved resistance to temperature and pH changes. Furthermore, the immobilized enzymes were reported to maintain activity after several use cycles. These improvements in the catalytic function of the immobilized enzymes have been attributed to a reduced mobility of the three-dimensional protein structure of the enzyme within nanofibers, thus decreasing the effect of the pH and the temperature in the denaturation of the enzyme. In addition, the immobilization of the enzyme through electrospinning is carried out in a
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single step, avoiding the exposure of the enzyme to various immobilization stages that can increase the opportunities of its denaturation [22, 25,26,30]. The main application of immobilized enzymes is in the fabrication of biosensors of high sensitivity and selectivity. A good example of this is the work of Ren et al. in the immobilization of the enzyme glucose oxidase (GOD) in poly(vinyl alcohol) (PVA) electrospun nanofibers and its use as a glucose biosensor in patients with diabetes [23]. Fructose dehydrogenase, horseradish peroxidase, urease and tyrosinase are additional examples of enzymes used for this purpose [24,27–29]. In most cases, the immobilization of the enzymes in electrospun nanofibers produced an improvement in the sensitivity and the selectivity of the biosensor. The high porosity and the elevated surface area of nanofibers can improve the interaction between the immobilized enzyme and the targeted analyte. The electrospun nanofibers have also been used in the fabrication of drug-release systems, including encapsulated enzymes. Here, the nanostructure of the nanofibers allows the modulation of the release pattern of the encapsulated enzyme. Moreover, the large superficial area allows an increase in the amount of the encapsulated protein. Previously, we reported the encapsulation and the subsequent release of the enzyme lactate dehydrogenase (LDH) encapsulated in nanofibers of
Fig. 1. SEM images of PVA nanofibers with different enzyme concentrations: (a) PVA without enzyme, (b) 5%, (c) 10%, (d) 16%, and (e) 33%.
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Fig. 2. Diameter distribution analysis of figures shown in Fig. 1.
PVA through the coaxial electrospinning technique. The encapsulated LDH showed different release patterns depending on the nature of the arrangement during the electrospun nanofiber formation, i.e., whether the enzyme was encapsulated in the core or immobilized on the
nanofiber surface [22]. Other uses of enzyme immobilization by electrospun nanofibers include biofuel cell applications, ethanol production, removal of pollutants from contaminated soil, sugar production from microalgae, antibacterial applications, etc. [25,26,30–32].
Fig. 3. SEM images of: (a) PVA nanofibers with 13% papain content (b) and PVA nanofibers after the glutaraldehyde vapor crosslinking process and their diameter distribution analysis ((c) and (d) respectively).
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2.3. Papain immobilization The immobilization of the papain encapsulated in the nanofibers was achieved by PVA-crosslinking using glutaraldehyde. This aldehyde is widely used as a crosslinking agent in enzyme immobilization [25, 33]. Typically, a PVA nanofiber mat was left in contact with glutaraldehyde vapor for various time periods inside a desiccator. After that, the samples were withdrawn from the desiccator and heat-treated at 40 °C in an oven for 20 min. Finally, the samples were stored at room temperature for 24 h, before their use in the enzymatic activity assays. 2.4. Determination of the encapsulated protein amount
Fig. 4. FTIR spectra of crude papain (a), PVA nanofibers (b), encapsulated papain within the PVA nanofibers (c) and the immobilized papain within the crosslinked PVA nanofibers (d).
In this work, we report the immobilization of the enzyme papain onto electrospun poly(vinyl alcohol) (PVA) nanofibers. The morphology of the electrospun PVA fibers loaded with papain is discussed along with the effect of enzyme loading efficiency, the crosslinking process and the reusability on the activity of immobilized protease.
The amount of encapsulated enzyme on the PVA membrane was measured by the Bradford protein assay. Samples were immersed overnight in a Tris–HCl buffer solution (pH 7.5). Under these conditions, the papain that is loose or not bounded to the nanofiber membrane is released to the buffer solution. The amount of papain released to the solution was measured following the Bradford method [34]. The enzyme loading efficiency was defined as the amount of papain (mg) in the membrane per milligram of the PVA nanofibrous membrane. 2.5. Enzyme activity measurement The amidase activity of the papain was measured using the Earlanger et al. method adapted to the papain [35], where Nα-
2. Materials and methods 2.1. Materials Papain (E.C. 3.4.22.2, 1.6 U/mg) from papaya (Carica papaya) latex, cysteine ≥ 97% (M w 121.16 g/mol), Nα-benzoyl- L -arginine 4-nitroanilide hydrochloride (BAPA) ≥ 98% (M w 434.88 g/mol), glutaraldehyde (GA, grade I, 50 wt.% aqueous solution), Tris buffer, acetic acid (ACS reagent ≥ 99.7%), bovine serum albumin (BSA) (lyophilized powder, ≥ 96% by agarose gel electrophoresis), PVA of high molecular weight and highly hydrolyzed (98% hydrolyzed, average Mw 126,000 g/mol), PVA of low molecular weight and partially hydrolyzed (87–89% hydrolyzed, average Mw 13,000–23,000 g/mol) and methanol were purchased from Sigma-Aldrich. Ethylenediaminetetraacetic acid (EDTA) (Mw 372.25 g/mol) and dimethyl sulfoxide (Mw 78.13) were supplied by Productos Químicos Monterrey. A Bradford protein assay kit was purchased from Biorad.
2.2. Papain encapsulation in electrospun PVA nanofibers The electrospinning setup consists of a syringe with a blunt metal needle, a high-voltage dc power supply (Spellman CZE1000R) and a grounded aluminum collector. A syringe pump (Cole Parmer) was used to control the feeding rates of the polymer solution. The encapsulated papain samples were prepared using a PVA solution of 8.0 wt.% with a 1:1 ratio of highly hydrolyzed and partially hydrolyzed PVA. The enzyme was added to the polymer solution until the targeted concentration of the enzyme was reached. These enzyme concentrations were calculated in relation with the polymer weight in each solution. Once the enzyme was dissolved in the polymer solution, the electrospinning process was carried out by applying a voltage of 20 kV, a feeding rate of 0.6 ml h−1 and an air gap of 15 cm between the grounded collector and the needle tip. Each batch of nanofiber mats was divided into four parts of equal weight. Two of these were utilized for the enzymatic activity assay, and the other two were used to calculate the amount of encapsulated protein.
Fig. 5. XPS analysis: a) wide-scan and b) N 1s core-level spectra of the crosslinked PVA nanofibers containing papain (red line) and the PVA nanofibers as control sample (black line).
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Table 1 Analysis by XPS. Sample
O 1s (%)
PVA membrane PVA/papain membrane
26.89 25.85
N 1s (%)
0.0 0.6
benzoyl-L-arginine 4-nitroanilide (BAPA) is used as a substrate. In a typical run, 1 ml of free-papain solution (with 1 mg ml−1) or an immobilized papain sample was placed into a test tube. Then, 5 ml of the activated substrate was added to each sample and the solution was left to react for 25 min. The enzymatic reaction was stopped by adding 1 ml of 30 wt.% acetic acid solution. The absorbance change at 410 nm, corresponding to the liberated p-nitroaniline, was measured spectrophotometrically. The enzymatic activity was expressed in BAPA units and was calculated following the next equation: BAPA units ¼ Δ410
nm = min
1000 3=8800:
The specific activity was obtained by normalizing the BAPA units with the amount of papain in the PVA nanofiber membrane. This specific activity was expressed in BAPA units per mg of protein. To study the effect of immobilization on the enzyme activity under storage, nanofibers containing encapsulated and immobilized papain were kept under refrigeration, and the activity was measured at various intervals for up to two weeks.
2.6. Characterization The morphology of the dried nanofibers was studied using a JSM7401F field emission scanning electron microscope (SEM). The UV–visible spectra were recorded using a SHIMADZU 2401 spectrophotometer. FTIR spectra were recorded in a Nexus 470 spectrophotometer. X-ray photoelectron spectroscopy (XPS) was carried out using an Axis Kratos Ultra operating at 10− 9 Torr level, with a monochromatic Al Ka ray source (15 mA, 15 mV). Transmission electron microscopy (TEM) studies were performed using a FEI TEM Titan G2 80–300 operated at 300 kV, with scanning-transmission electron microscopy (STEM) capabilities and equipped with a high angle annular dark field (HAADF) detector from Fishione, a bright field (BF) STEM detector from Gatan, an annular dark field (ADF) STEM detector from Gatan, and an EDAX energy dispersive X-ray spectroscopy (EDXS) detector.
C 1s (%)
284.8 eV (%)
286.4 eV (%)
288.9 eV (%)
73.03 73.31
44.43 48.36
46.92 42.63
8.65 9.02
3. Results and discussion 3.1. Morphology Highly hydrolyzed PVA is difficult to electrospin due to its high surface tension. One way to electrospin this polymer is by adding surfactants to lower its aqueous solution surface tension [36]. However, the formation of surfactant/papain complexes could affect the enzyme activity, and this approach was not followed. Instead, we used a different approach by blending partially hydrolyzed and low molecular weight PVA with highly hydrolyzed and larger molecular weight PVA. We think that the first polymer provides good mechanical properties and stability to the solvent, whereas the second polymer improves the electrospinnability of the enzyme/polymer solution. Fig. 1 shows the effect of the enzyme concentration on the final morphology of the electrospun nanofibers. Our approach to produce PVA nanofibers was successful because the nanofiber diameter distribution was not bimodal, which could indicate polymer phase separation (Fig. 2). PVA nanofibers without the enzyme (Fig. 1a) showed a defect-free morphology and an average diameter of 81 nm (Fig. 2a). Once the enzyme was encapsulated in the nanofibers, the nanofiber diameter increased up to 161 nm (Fig. 2(b–e)) with the presence of entangled fibers (see the white arrows in Fig. 1). The fiber diameter increment and the fiber entanglement are the expected morphology variations in electrospun nanofibers from concentrated poly(electrolyte) solutions [37]. Because the proteins are natural poly(electrolytes), this morphology has been observed in enzyme-loaded electrospun nanofibers [38]. The bead defects in some nanofibers could be attributed to a higher enzyme concentration in those particular areas (see white circles in Fig. 1e). In Fig. 3, we can observe the effect of crosslinking on the morphology of the electrospun nanofibers of PVA. Fig. 3a shows the PVA nanofibers with 13% of papain before any crosslinking treatment have a defect free morphology and an average diameter of 116 nm (Fig. 3c). After GAvt, the diameter of PVA/papain nanofibers increased their average diameter up to 174 nm (Fig. 3d), but still kept most of their fibrous morphology (Fig. 3b). The diameter increment could be related to the absorption of GA vapor inside the nanofiber structure. Nevertheless, the diameter increment was not appreciable in comparison to the diameter of the fibers without any crosslinking treatment. Hence, we can conclude that
Fig. 6. a) HAADF image of one of the nanofibers, where the red arrow indicates the region analyzed during the EDXS line scan. b) EDXS line scan profiles, showing the variations of the C Kα, O Kα and S Kα signals along the region analyzed.
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glutaraldehyde vapor treatment is an effective method to crosslink PVA nanofibers
3.2. Presence of papain in the PVA nanofibers FTIR and XPS analyses were used to prove the presence of the papain in the electrospun PVA nanofibers. In Fig. 4a we can observe the absorption band characteristic of free papain, the band at 1652 cm−1, corresponding to the C_0 stretching in the amide-I region, and the absorption band at 1540 cm− 1 of the amide-II region, attributed to N\H bending and C\N stretching [39]. The peak at 1090 cm−1 in the PVA nanofibers without enzyme is due to C\O stretching, and the stretching band at approximately 3315 cm−1 in the PVA nanofibers is associated with hydrogen-bonded alcohol (O\H) (Fig. 4b). In the IR spectrum of PVA nanofibers loaded with papain, the characteristic absorption bands of amide-I at 1650 cm−1 and amide-II at 1545 cm− 1 suggest that the papain is present in the PVA nanofibers (Fig. 4c). Moreover, in the IR spectrum of PVA nanofibers loaded with papain, the absorption band at 3315 cm−1 was wider than that in the PVA nanofibers alone (Fig. 4c). This change has been attributed to the superposition of the stretching vibration of OH and N\H. The previously discussed absorption bands are characteristic of proteins, which is also indicative of the presence of papain in the PVA nanofibers [40–42]. In the IR spectrum of the crosslinked samples (Fig. 4d), the presence of glutaraldehyde was confirmed by the presence of typical absorption bands at 1100 cm−1 for the aliphatic groups of glutaraldehyde and the absorption band at 1720 cm−1 for the free aldehyde groups. Additionally, the crosslinking process was confirmed by the presence of the absorption band at 1150 cm−1, which indicates the formation of \C\O\C\ bonds, generated by a reaction between the \OH group of a PVA molecule and the \C\ of a glutaraldehyde molecule [43]. The characteristic bands of the immobilized papain at 1653 cm−1 for amide-I and at 1547 cm−1 for amide-II were quite similar for free papain and the encapsulated papain. The previous results strongly suggest that the three-dimensional structure of papain was conserved during and after the immobilization process. The surface chemistry composition of PVA and the PVA/papain membranes was analyzed by XPS (Fig. 5). Here, nitrogen is observed in the PVA/papain membrane, which indicates the presence of papain, whereas this element is absent in the control PVA non-woven nanofiber membrane. During C 1s deconvolution, three components (284.8, 286.4, and 288.9 eV) were proposed. The first component corresponds mainly to a C\C signal, which is close to 50%, as is expected for PVA, whereas the other two, at 286.4 eV and 288.9 eV, are assigned to C\O and O_C\O signals, respectively.
The concentration percentages (%) of these components are shown in Table 1. The concentration at 288.9 eV is higher in the PVA/papain membrane sample. This can be explained by the fact that the enzymes have many carboxyl groups in their proteic structure. The 0.6% mass of nitrogen indicates a 3.75% mass of protein, assuming 16% nitrogen content in papain. However, the amounts of nitrogen and carboxylic group, which are related to the amount of enzyme, were not as high as anticipated. This information indicates that the papain molecules were not only attached onto the surface of the electrospun fibers, but they were also embedded within the PVA nanofibers [42,44,45]. Both the IR and XPS analyses verified that papain was immobilized in PVA nanofibers. An EDXS analysis was performed on the nanofibers to obtain a better insight into the elemental distribution within them. Fig. 6 shows a highangle annular dark field (HAADF) image of one of the nanofibers (Fig. 6a), where the red line indicates the region analyzed and the direction of analysis. Fig. 6b shows the EDXS line scan profile where the signals correspond to the intensity variations in the C Kα, O Kα and S Kα energies. The profile of the last element shows three clear peaks along the line scan. It is well known that sulphur is not present in the molecular structure of PVA, so we assume that a sulphur signal is related with the cysteine residues present in the papain immobilized on the PVA nanofibers. The regions showing a higher sulphur signal could be associated to areas that have a higher amount of enzyme. In this manner, we were able to obtain a sulphur distribution profile across the width of the
Fig. 7. Effect of the crosslinking process in the enzyme activity of the immobilized papain samples.
Fig. 9. Relative activity of the immobilized papain after several cycles of reuse.
Fig. 8. Effect of the concentration of papain solution on the loading efficiency and the retained specific activity of the immobilized papain samples.
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Fig. 10. Effect of the reuse cycles in the nanofiber morphology: a) first cycle, b) second cycle, c) third cycle, d) fourth cycle, e) fifth cycle, f) sixth cycle.
nanofiber. This profile suggests that the protein is present in the center and the edges of the nanofibers.
vapor, the maximum enzymatic activity for papain was reached after 30 min of exposure (Fig. 7).
3.3. Effect of the crosslinking time 3.4. Enzymatic activity of the immobilized papain It is well known that the crosslinking reaction modifies the activity of immobilized enzymes because it can affect the accessibility of the substrate to the active site of the enzyme or even cause its complete inactivation [42]. GA was chosen as a crosslinking agent due to its high reactivity toward the amine and hydroxyl groups. Once the encapsulated enzyme in PVA nanofibers was left in contact with the glutaraldehyde
Fig. 11. Analysis of the effect of the storage time on the enzymatic activity of the immobilized papain.
The effects of the concentration of the papain solution on the loading efficiency and the retained specific activity of the immobilized papain in PVA membranes are shown in Fig. 8. Although the percentage of the papain loading into the PVA membranes showed a steady increment, the retained specific activity reached a maximum peak at 13% papain content. At this point, the loading efficiency is approximately 15%, and even when an increment of the concentration of papain solution improves the loading efficiency, there is a sharp drop in the catalytic activity. This observation could be related to the fact that the enzymes are now confined into a solid matrix, and as a consequence, the access to the substrate is now more restricted compared to that for the free enzyme. Nevertheless, it is unlikely that the enzyme molecules present in the polymer solution were immobilized on the surface of the nanofibers. Thereby, not all of the enzyme molecules are actually exposed to the substrate to participate in the reaction [42]. Hence, the decrease in the enzymatic activity can be explained due to the high amount of encapsulated papain during the electrospinning process. This factor, in combination with the nanofiber crosslinking reaction, could limit the diffusion of the substrate to the active site of the immobilized enzyme [33,42]. In this study, the maximum enzymatic activity reached by the papain immobilized in electrospun PVA membranes was approximately 88% that of the free papain (1.22 × 10−2 U/mg). This value is higher than several that have been reported in the immobilization and stabilization of papain using different immobilization supports [46–49]. The enzymatic activity of papain was preserved after immobilization, whereas it was not for the free enzyme; this suggests that the threedimensional structure of papain was not affected. The enzymatic activity of papain preserved after immobilization as compared to the free enzyme suggests that the three-dimensional structure of papain was not affected, either by the electrostatic forces involved in the
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electrospinning process, or by the polar intermolecular interactions with the PVA.
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are greatly appreciated. The authors also want to thank Ms. Gabriela Padrón and M.C. Mónica Ceniceros for their technical assistance.
3.5. Reusability of the immobilized papain References The reusability of immobilized papain was analyzed by measuring the activity during several cycles. As we can observe in Fig. 9, the relative activity of the immobilized papain decreased along with the reusing cycles. The immobilized enzyme retained over 12% of its initial activity after six cycles of reuse. Such reusability could be advantageous for the continuous use of the immobilized enzyme in industrial applications. It is likely that enzymatic activity loss is in some way related with the following reasons. First, it is expected that, during the immobilization, not all of the enzyme molecules would be joined to the PVA nanofibers. Second, the loose fibers could be released from the membrane after each reuse cycle [42]. Finally, as we can see in Fig. 10 after several reuse cycles it is possible that the hydrophilic nature of the PVA chains that are not crosslinked could cause a gradual loss of the fibrous morphology of the PVA membranes, reducing the total surface area of the nanofiber membranes, as well lowering the effectiveness of the enzymatic function of the immobilized papain [25,42]. 3.6. Effect of storage time on the enzymatic activity The analysis of the effect of the storage time on the enzymatic activity of the immobilized papain in PVA nanofibers was also analyzed (Fig. 11). The catalytic behavior of the immobilized papain was much better than that of the free enzyme. Also, the immobilized papain retains nearly 40% of its initial activity after 14 days of storage. The intermolecular bonds between papain and the PVA nanofibers, formed by the crosslinking reaction with GA, apparently has a positive effect during the storage time on the catalytic behavior of the immobilized papain [25,42]. 4. Conclusions Papain (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in PVA nanofibers by the electrospinning technique. By blending in a solution of PVA of various molecular weights (partially and highly hydrolyzed polymers), it was possible to electrospin the polymer without using any organic solvent, surfactant or low molecular weight additives. The morphology of the PVA nanofibers was analyzed by SEM, and it was shown that at low and moderate enzyme concentrations, there was no drastic change in the morphology of the nanofibers. The presence of papain in the electrospun nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The retained activity of the immobilized papain in the electrospun PVA nanofiber mat after a crosslinking reaction with glutaraldehyde vapor was approximately 88% relative to the free enzyme. The optimum crosslinking time was 30 min. The reusability of the immobilized enzyme was confirmed by the presence of residual activity after six cycles of reuse. Furthermore, the immobilized enzyme retains 40% of the initial activity after 14 days of storage. This work provides evidence that supports electrospinning as an effective and versatile technique for the encapsulation and immobilization of enzymes in polymer nanofibers. The known sensibility of papain toward metal ions as Hg2 + presents the possibility of its application in the fabrication of biosensors specialized in the detection of metal ions. Acknowledgments The funding support from CONACYT-Mexico (Project 174806, Convocatoria conjunta CONACYT-CNPq Mexico–Brasil en nanotecnologías 2011) and CONACYT (Project 176671, Convocatoria CienciaBásica 2012) as well as PROMEP-SEP-Mexico (103.5/12/4296)
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Encapsulation and immobilization of papain in electrospun nanofibrous membranes of PVA cross-linked with glutaraldehyde vapor Iván E. Moreno-Cortez a,b,c, Jorge Romero-García a,⁎, Virgilio González-González b,c, Domingo I. García-Gutierrez b,c, Marco A. Garza-Navarro b,c, Rodolfo Cruz-Silva d a
Centro de Investigación en Química Aplicada (CIQA), Blvd. Enrique Reyna # 140, San José de los Cerritos, Saltillo, Coahuila 25100, México Universidad Autónoma de Nuevo León (UANL), Fac. de Ingeniería Mecánica y Eléctrica (FIME), Av. Universidad S/N, Cd. Universitaria, San Nicolás de los Garza, Nuevo León C.P. 66450, México Universidad Autónoma de Nuevo León (UANL), Centro de Innovación, Investigación y Desarrollo en Ingeniería y Tecnología (CIIDIT), Apodaca, Nuevo León, México d Research Center for Exotic Nanocarbons, Shinshu University, 4-17-1 Wakasato, Nagano 380-8553, Japan b c
a r t i c l e
i n f o
Article history: Received 12 January 2015 Received in revised form 12 March 2015 Accepted 23 March 2015 Available online 25 March 2015 Keywords: Nanofibers Papain Enzyme immobilization Electrospinning
a b s t r a c t In this paper, papain enzyme (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in poly(vinyl alcohol) (PVA) nanofibers prepared by electrospinning. The morphology of the electrospun nanofibers was characterized by scanning electron microscopy (SEM) and the diameter distribution was in the range of 80 to 170 nm. The presence of the enzyme within the PVA nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The maximum catalytic activity was reached when the enzyme loading was 13%. The immobilization of papain in the nanofiber membrane was achieved by chemical crosslinking with a glutaraldehyde vapor treatment (GAvt). The catalytic activity of the immobilized papain was 88% with respect to the free enzyme. The crosslinking time by GAvt to immobilize the enzyme onto the nanofiber mat was 24 h, and the enzyme retained its catalytic activity after six cycles. The crosslinked samples maintained 40% of their initial activity after being stored for 14 days. PVA electrospun nanofibers are excellent matrices for the immobilization of enzymes due to their high surface area and their nanoporous structure. © 2015 Published by Elsevier B.V.
1. Introduction The immobilization of enzymes has evolved into a common technique to extend the catalytic activity of these important biological molecules that usually lose their three-dimensional molecular structure when they are in solution. Papain is a thiol protease commonly found in many organisms, such as plants and fungi. The kinetics and the structure of papain have been well-studied; it is considered the enzyme par excellence to compare the efficiency of various immobilization methods [1]. The applications of papain in many industrial, medical, and pharmaceutical technologies have fostered research to develop a variety of immobilization protocols [2]. This protease has been extensively used in traditional food processing applications as a chill-proofing agent: for instance, during beer-finishing operations in the brewing process and also to tenderize meat in the meat industry [3,4]. Papain has been used as a catalyst to oligomerize hydrophobic amino acids [5–11]. The potential uses of papain include the development of assays and biosensors. For instance, the strong interaction between bioconjugated carbon nanotubes and papain opens up the possibility of biosensors using these materials for the detection of 9-carboxy tetrahydrocannabinol in urine ⁎ Corresponding author. E-mail address: [email protected] (J. Romero-García).
http://dx.doi.org/10.1016/j.msec.2015.03.049 0928-4931/© 2015 Published by Elsevier B.V.
to test marijuana consumption [12]. Kim et al. created a modulated fluorescent sensor, in which Hg+ induced the agglutination of a poly(paraphenyleneethynylene)–papain complex [13]. Recently, several nanomaterials have been used as a matrix for papain immobilization. Among them, we can find magnetic nanoparticles [14], carbon nanotubes [15] and polymeric micro- and nanoparticles [2,16]. A different reported method demonstrated that cross-linked enzyme aggregates (CLEAs) represent a simple and useful enzyme immobilization technique, which was recently applied in the immobilization of papain [17]. Another technique, known as electrospinning, has been used in the immobilization of several enzymes [22] but not for the immobilization of papain. A great deal of information regarding electrospun polymer fibers and their applications can be found in the literature [18]. The process of nanofiber formation by electrospinning can be successfully controlled to obtain long and uniform nanofibers, which can be deposited to form non-woven mats and membranes with open porosity, high surface area and good mechanical properties. Several research groups around the world have used electrospinning in the immobilization of biologically active molecules and cells of commercial and scientific relevance, such as viruses, bacteria and various types of cells that have successfully been immobilized on polymeric electrospun membranes [19,20]. The encapsulated biomolecules have
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been used in important and interesting applications, such as biosensors, drug release systems and tissue engineering [20–22]. The enzymes have a fundamental role in the structure and metabolism of the human body in order to keep cells with optimal performance. Moreover, enzymes are important biomolecules in scientific research, as well as in industrial processes. A variety of enzymes has been encapsulated and immobilized using the electrospinning technique. A few examples are: glucose oxidase, lipase, laccase, lysozyme, lactate dehydrogenase, horseradish peroxidase, fructose dehydrogenase, urease, tyrosinase and cellulase [22–30]. In most of the reported cases of enzyme immobilization using electrospinning, it has been demonstrated that neither the electric field nor the polymer–enzyme interactions present in the electrospinning process affect the catalytic function of the immobilized enzymes. In general, the catalytic function of the immobilized enzymes presents an improved resistance to temperature and pH changes. Furthermore, the immobilized enzymes were reported to maintain activity after several use cycles. These improvements in the catalytic function of the immobilized enzymes have been attributed to a reduced mobility of the three-dimensional protein structure of the enzyme within nanofibers, thus decreasing the effect of the pH and the temperature in the denaturation of the enzyme. In addition, the immobilization of the enzyme through electrospinning is carried out in a
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single step, avoiding the exposure of the enzyme to various immobilization stages that can increase the opportunities of its denaturation [22, 25,26,30]. The main application of immobilized enzymes is in the fabrication of biosensors of high sensitivity and selectivity. A good example of this is the work of Ren et al. in the immobilization of the enzyme glucose oxidase (GOD) in poly(vinyl alcohol) (PVA) electrospun nanofibers and its use as a glucose biosensor in patients with diabetes [23]. Fructose dehydrogenase, horseradish peroxidase, urease and tyrosinase are additional examples of enzymes used for this purpose [24,27–29]. In most cases, the immobilization of the enzymes in electrospun nanofibers produced an improvement in the sensitivity and the selectivity of the biosensor. The high porosity and the elevated surface area of nanofibers can improve the interaction between the immobilized enzyme and the targeted analyte. The electrospun nanofibers have also been used in the fabrication of drug-release systems, including encapsulated enzymes. Here, the nanostructure of the nanofibers allows the modulation of the release pattern of the encapsulated enzyme. Moreover, the large superficial area allows an increase in the amount of the encapsulated protein. Previously, we reported the encapsulation and the subsequent release of the enzyme lactate dehydrogenase (LDH) encapsulated in nanofibers of
Fig. 1. SEM images of PVA nanofibers with different enzyme concentrations: (a) PVA without enzyme, (b) 5%, (c) 10%, (d) 16%, and (e) 33%.
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Fig. 2. Diameter distribution analysis of figures shown in Fig. 1.
PVA through the coaxial electrospinning technique. The encapsulated LDH showed different release patterns depending on the nature of the arrangement during the electrospun nanofiber formation, i.e., whether the enzyme was encapsulated in the core or immobilized on the
nanofiber surface [22]. Other uses of enzyme immobilization by electrospun nanofibers include biofuel cell applications, ethanol production, removal of pollutants from contaminated soil, sugar production from microalgae, antibacterial applications, etc. [25,26,30–32].
Fig. 3. SEM images of: (a) PVA nanofibers with 13% papain content (b) and PVA nanofibers after the glutaraldehyde vapor crosslinking process and their diameter distribution analysis ((c) and (d) respectively).
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2.3. Papain immobilization The immobilization of the papain encapsulated in the nanofibers was achieved by PVA-crosslinking using glutaraldehyde. This aldehyde is widely used as a crosslinking agent in enzyme immobilization [25, 33]. Typically, a PVA nanofiber mat was left in contact with glutaraldehyde vapor for various time periods inside a desiccator. After that, the samples were withdrawn from the desiccator and heat-treated at 40 °C in an oven for 20 min. Finally, the samples were stored at room temperature for 24 h, before their use in the enzymatic activity assays. 2.4. Determination of the encapsulated protein amount
Fig. 4. FTIR spectra of crude papain (a), PVA nanofibers (b), encapsulated papain within the PVA nanofibers (c) and the immobilized papain within the crosslinked PVA nanofibers (d).
In this work, we report the immobilization of the enzyme papain onto electrospun poly(vinyl alcohol) (PVA) nanofibers. The morphology of the electrospun PVA fibers loaded with papain is discussed along with the effect of enzyme loading efficiency, the crosslinking process and the reusability on the activity of immobilized protease.
The amount of encapsulated enzyme on the PVA membrane was measured by the Bradford protein assay. Samples were immersed overnight in a Tris–HCl buffer solution (pH 7.5). Under these conditions, the papain that is loose or not bounded to the nanofiber membrane is released to the buffer solution. The amount of papain released to the solution was measured following the Bradford method [34]. The enzyme loading efficiency was defined as the amount of papain (mg) in the membrane per milligram of the PVA nanofibrous membrane. 2.5. Enzyme activity measurement The amidase activity of the papain was measured using the Earlanger et al. method adapted to the papain [35], where Nα-
2. Materials and methods 2.1. Materials Papain (E.C. 3.4.22.2, 1.6 U/mg) from papaya (Carica papaya) latex, cysteine ≥ 97% (M w 121.16 g/mol), Nα-benzoyl- L -arginine 4-nitroanilide hydrochloride (BAPA) ≥ 98% (M w 434.88 g/mol), glutaraldehyde (GA, grade I, 50 wt.% aqueous solution), Tris buffer, acetic acid (ACS reagent ≥ 99.7%), bovine serum albumin (BSA) (lyophilized powder, ≥ 96% by agarose gel electrophoresis), PVA of high molecular weight and highly hydrolyzed (98% hydrolyzed, average Mw 126,000 g/mol), PVA of low molecular weight and partially hydrolyzed (87–89% hydrolyzed, average Mw 13,000–23,000 g/mol) and methanol were purchased from Sigma-Aldrich. Ethylenediaminetetraacetic acid (EDTA) (Mw 372.25 g/mol) and dimethyl sulfoxide (Mw 78.13) were supplied by Productos Químicos Monterrey. A Bradford protein assay kit was purchased from Biorad.
2.2. Papain encapsulation in electrospun PVA nanofibers The electrospinning setup consists of a syringe with a blunt metal needle, a high-voltage dc power supply (Spellman CZE1000R) and a grounded aluminum collector. A syringe pump (Cole Parmer) was used to control the feeding rates of the polymer solution. The encapsulated papain samples were prepared using a PVA solution of 8.0 wt.% with a 1:1 ratio of highly hydrolyzed and partially hydrolyzed PVA. The enzyme was added to the polymer solution until the targeted concentration of the enzyme was reached. These enzyme concentrations were calculated in relation with the polymer weight in each solution. Once the enzyme was dissolved in the polymer solution, the electrospinning process was carried out by applying a voltage of 20 kV, a feeding rate of 0.6 ml h−1 and an air gap of 15 cm between the grounded collector and the needle tip. Each batch of nanofiber mats was divided into four parts of equal weight. Two of these were utilized for the enzymatic activity assay, and the other two were used to calculate the amount of encapsulated protein.
Fig. 5. XPS analysis: a) wide-scan and b) N 1s core-level spectra of the crosslinked PVA nanofibers containing papain (red line) and the PVA nanofibers as control sample (black line).
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Table 1 Analysis by XPS. Sample
O 1s (%)
PVA membrane PVA/papain membrane
26.89 25.85
N 1s (%)
0.0 0.6
benzoyl-L-arginine 4-nitroanilide (BAPA) is used as a substrate. In a typical run, 1 ml of free-papain solution (with 1 mg ml−1) or an immobilized papain sample was placed into a test tube. Then, 5 ml of the activated substrate was added to each sample and the solution was left to react for 25 min. The enzymatic reaction was stopped by adding 1 ml of 30 wt.% acetic acid solution. The absorbance change at 410 nm, corresponding to the liberated p-nitroaniline, was measured spectrophotometrically. The enzymatic activity was expressed in BAPA units and was calculated following the next equation: BAPA units ¼ Δ410
nm = min
1000 3=8800:
The specific activity was obtained by normalizing the BAPA units with the amount of papain in the PVA nanofiber membrane. This specific activity was expressed in BAPA units per mg of protein. To study the effect of immobilization on the enzyme activity under storage, nanofibers containing encapsulated and immobilized papain were kept under refrigeration, and the activity was measured at various intervals for up to two weeks.
2.6. Characterization The morphology of the dried nanofibers was studied using a JSM7401F field emission scanning electron microscope (SEM). The UV–visible spectra were recorded using a SHIMADZU 2401 spectrophotometer. FTIR spectra were recorded in a Nexus 470 spectrophotometer. X-ray photoelectron spectroscopy (XPS) was carried out using an Axis Kratos Ultra operating at 10− 9 Torr level, with a monochromatic Al Ka ray source (15 mA, 15 mV). Transmission electron microscopy (TEM) studies were performed using a FEI TEM Titan G2 80–300 operated at 300 kV, with scanning-transmission electron microscopy (STEM) capabilities and equipped with a high angle annular dark field (HAADF) detector from Fishione, a bright field (BF) STEM detector from Gatan, an annular dark field (ADF) STEM detector from Gatan, and an EDAX energy dispersive X-ray spectroscopy (EDXS) detector.
C 1s (%)
284.8 eV (%)
286.4 eV (%)
288.9 eV (%)
73.03 73.31
44.43 48.36
46.92 42.63
8.65 9.02
3. Results and discussion 3.1. Morphology Highly hydrolyzed PVA is difficult to electrospin due to its high surface tension. One way to electrospin this polymer is by adding surfactants to lower its aqueous solution surface tension [36]. However, the formation of surfactant/papain complexes could affect the enzyme activity, and this approach was not followed. Instead, we used a different approach by blending partially hydrolyzed and low molecular weight PVA with highly hydrolyzed and larger molecular weight PVA. We think that the first polymer provides good mechanical properties and stability to the solvent, whereas the second polymer improves the electrospinnability of the enzyme/polymer solution. Fig. 1 shows the effect of the enzyme concentration on the final morphology of the electrospun nanofibers. Our approach to produce PVA nanofibers was successful because the nanofiber diameter distribution was not bimodal, which could indicate polymer phase separation (Fig. 2). PVA nanofibers without the enzyme (Fig. 1a) showed a defect-free morphology and an average diameter of 81 nm (Fig. 2a). Once the enzyme was encapsulated in the nanofibers, the nanofiber diameter increased up to 161 nm (Fig. 2(b–e)) with the presence of entangled fibers (see the white arrows in Fig. 1). The fiber diameter increment and the fiber entanglement are the expected morphology variations in electrospun nanofibers from concentrated poly(electrolyte) solutions [37]. Because the proteins are natural poly(electrolytes), this morphology has been observed in enzyme-loaded electrospun nanofibers [38]. The bead defects in some nanofibers could be attributed to a higher enzyme concentration in those particular areas (see white circles in Fig. 1e). In Fig. 3, we can observe the effect of crosslinking on the morphology of the electrospun nanofibers of PVA. Fig. 3a shows the PVA nanofibers with 13% of papain before any crosslinking treatment have a defect free morphology and an average diameter of 116 nm (Fig. 3c). After GAvt, the diameter of PVA/papain nanofibers increased their average diameter up to 174 nm (Fig. 3d), but still kept most of their fibrous morphology (Fig. 3b). The diameter increment could be related to the absorption of GA vapor inside the nanofiber structure. Nevertheless, the diameter increment was not appreciable in comparison to the diameter of the fibers without any crosslinking treatment. Hence, we can conclude that
Fig. 6. a) HAADF image of one of the nanofibers, where the red arrow indicates the region analyzed during the EDXS line scan. b) EDXS line scan profiles, showing the variations of the C Kα, O Kα and S Kα signals along the region analyzed.
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glutaraldehyde vapor treatment is an effective method to crosslink PVA nanofibers
3.2. Presence of papain in the PVA nanofibers FTIR and XPS analyses were used to prove the presence of the papain in the electrospun PVA nanofibers. In Fig. 4a we can observe the absorption band characteristic of free papain, the band at 1652 cm−1, corresponding to the C_0 stretching in the amide-I region, and the absorption band at 1540 cm− 1 of the amide-II region, attributed to N\H bending and C\N stretching [39]. The peak at 1090 cm−1 in the PVA nanofibers without enzyme is due to C\O stretching, and the stretching band at approximately 3315 cm−1 in the PVA nanofibers is associated with hydrogen-bonded alcohol (O\H) (Fig. 4b). In the IR spectrum of PVA nanofibers loaded with papain, the characteristic absorption bands of amide-I at 1650 cm−1 and amide-II at 1545 cm− 1 suggest that the papain is present in the PVA nanofibers (Fig. 4c). Moreover, in the IR spectrum of PVA nanofibers loaded with papain, the absorption band at 3315 cm−1 was wider than that in the PVA nanofibers alone (Fig. 4c). This change has been attributed to the superposition of the stretching vibration of OH and N\H. The previously discussed absorption bands are characteristic of proteins, which is also indicative of the presence of papain in the PVA nanofibers [40–42]. In the IR spectrum of the crosslinked samples (Fig. 4d), the presence of glutaraldehyde was confirmed by the presence of typical absorption bands at 1100 cm−1 for the aliphatic groups of glutaraldehyde and the absorption band at 1720 cm−1 for the free aldehyde groups. Additionally, the crosslinking process was confirmed by the presence of the absorption band at 1150 cm−1, which indicates the formation of \C\O\C\ bonds, generated by a reaction between the \OH group of a PVA molecule and the \C\ of a glutaraldehyde molecule [43]. The characteristic bands of the immobilized papain at 1653 cm−1 for amide-I and at 1547 cm−1 for amide-II were quite similar for free papain and the encapsulated papain. The previous results strongly suggest that the three-dimensional structure of papain was conserved during and after the immobilization process. The surface chemistry composition of PVA and the PVA/papain membranes was analyzed by XPS (Fig. 5). Here, nitrogen is observed in the PVA/papain membrane, which indicates the presence of papain, whereas this element is absent in the control PVA non-woven nanofiber membrane. During C 1s deconvolution, three components (284.8, 286.4, and 288.9 eV) were proposed. The first component corresponds mainly to a C\C signal, which is close to 50%, as is expected for PVA, whereas the other two, at 286.4 eV and 288.9 eV, are assigned to C\O and O_C\O signals, respectively.
The concentration percentages (%) of these components are shown in Table 1. The concentration at 288.9 eV is higher in the PVA/papain membrane sample. This can be explained by the fact that the enzymes have many carboxyl groups in their proteic structure. The 0.6% mass of nitrogen indicates a 3.75% mass of protein, assuming 16% nitrogen content in papain. However, the amounts of nitrogen and carboxylic group, which are related to the amount of enzyme, were not as high as anticipated. This information indicates that the papain molecules were not only attached onto the surface of the electrospun fibers, but they were also embedded within the PVA nanofibers [42,44,45]. Both the IR and XPS analyses verified that papain was immobilized in PVA nanofibers. An EDXS analysis was performed on the nanofibers to obtain a better insight into the elemental distribution within them. Fig. 6 shows a highangle annular dark field (HAADF) image of one of the nanofibers (Fig. 6a), where the red line indicates the region analyzed and the direction of analysis. Fig. 6b shows the EDXS line scan profile where the signals correspond to the intensity variations in the C Kα, O Kα and S Kα energies. The profile of the last element shows three clear peaks along the line scan. It is well known that sulphur is not present in the molecular structure of PVA, so we assume that a sulphur signal is related with the cysteine residues present in the papain immobilized on the PVA nanofibers. The regions showing a higher sulphur signal could be associated to areas that have a higher amount of enzyme. In this manner, we were able to obtain a sulphur distribution profile across the width of the
Fig. 7. Effect of the crosslinking process in the enzyme activity of the immobilized papain samples.
Fig. 9. Relative activity of the immobilized papain after several cycles of reuse.
Fig. 8. Effect of the concentration of papain solution on the loading efficiency and the retained specific activity of the immobilized papain samples.
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Fig. 10. Effect of the reuse cycles in the nanofiber morphology: a) first cycle, b) second cycle, c) third cycle, d) fourth cycle, e) fifth cycle, f) sixth cycle.
nanofiber. This profile suggests that the protein is present in the center and the edges of the nanofibers.
vapor, the maximum enzymatic activity for papain was reached after 30 min of exposure (Fig. 7).
3.3. Effect of the crosslinking time 3.4. Enzymatic activity of the immobilized papain It is well known that the crosslinking reaction modifies the activity of immobilized enzymes because it can affect the accessibility of the substrate to the active site of the enzyme or even cause its complete inactivation [42]. GA was chosen as a crosslinking agent due to its high reactivity toward the amine and hydroxyl groups. Once the encapsulated enzyme in PVA nanofibers was left in contact with the glutaraldehyde
Fig. 11. Analysis of the effect of the storage time on the enzymatic activity of the immobilized papain.
The effects of the concentration of the papain solution on the loading efficiency and the retained specific activity of the immobilized papain in PVA membranes are shown in Fig. 8. Although the percentage of the papain loading into the PVA membranes showed a steady increment, the retained specific activity reached a maximum peak at 13% papain content. At this point, the loading efficiency is approximately 15%, and even when an increment of the concentration of papain solution improves the loading efficiency, there is a sharp drop in the catalytic activity. This observation could be related to the fact that the enzymes are now confined into a solid matrix, and as a consequence, the access to the substrate is now more restricted compared to that for the free enzyme. Nevertheless, it is unlikely that the enzyme molecules present in the polymer solution were immobilized on the surface of the nanofibers. Thereby, not all of the enzyme molecules are actually exposed to the substrate to participate in the reaction [42]. Hence, the decrease in the enzymatic activity can be explained due to the high amount of encapsulated papain during the electrospinning process. This factor, in combination with the nanofiber crosslinking reaction, could limit the diffusion of the substrate to the active site of the immobilized enzyme [33,42]. In this study, the maximum enzymatic activity reached by the papain immobilized in electrospun PVA membranes was approximately 88% that of the free papain (1.22 × 10−2 U/mg). This value is higher than several that have been reported in the immobilization and stabilization of papain using different immobilization supports [46–49]. The enzymatic activity of papain was preserved after immobilization, whereas it was not for the free enzyme; this suggests that the threedimensional structure of papain was not affected. The enzymatic activity of papain preserved after immobilization as compared to the free enzyme suggests that the three-dimensional structure of papain was not affected, either by the electrostatic forces involved in the
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electrospinning process, or by the polar intermolecular interactions with the PVA.
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are greatly appreciated. The authors also want to thank Ms. Gabriela Padrón and M.C. Mónica Ceniceros for their technical assistance.
3.5. Reusability of the immobilized papain References The reusability of immobilized papain was analyzed by measuring the activity during several cycles. As we can observe in Fig. 9, the relative activity of the immobilized papain decreased along with the reusing cycles. The immobilized enzyme retained over 12% of its initial activity after six cycles of reuse. Such reusability could be advantageous for the continuous use of the immobilized enzyme in industrial applications. It is likely that enzymatic activity loss is in some way related with the following reasons. First, it is expected that, during the immobilization, not all of the enzyme molecules would be joined to the PVA nanofibers. Second, the loose fibers could be released from the membrane after each reuse cycle [42]. Finally, as we can see in Fig. 10 after several reuse cycles it is possible that the hydrophilic nature of the PVA chains that are not crosslinked could cause a gradual loss of the fibrous morphology of the PVA membranes, reducing the total surface area of the nanofiber membranes, as well lowering the effectiveness of the enzymatic function of the immobilized papain [25,42]. 3.6. Effect of storage time on the enzymatic activity The analysis of the effect of the storage time on the enzymatic activity of the immobilized papain in PVA nanofibers was also analyzed (Fig. 11). The catalytic behavior of the immobilized papain was much better than that of the free enzyme. Also, the immobilized papain retains nearly 40% of its initial activity after 14 days of storage. The intermolecular bonds between papain and the PVA nanofibers, formed by the crosslinking reaction with GA, apparently has a positive effect during the storage time on the catalytic behavior of the immobilized papain [25,42]. 4. Conclusions Papain (E.C. 3.4.22.2, 1.6 U/mg) was successfully immobilized in PVA nanofibers by the electrospinning technique. By blending in a solution of PVA of various molecular weights (partially and highly hydrolyzed polymers), it was possible to electrospin the polymer without using any organic solvent, surfactant or low molecular weight additives. The morphology of the PVA nanofibers was analyzed by SEM, and it was shown that at low and moderate enzyme concentrations, there was no drastic change in the morphology of the nanofibers. The presence of papain in the electrospun nanofibers was confirmed by infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDXS) analyses. The retained activity of the immobilized papain in the electrospun PVA nanofiber mat after a crosslinking reaction with glutaraldehyde vapor was approximately 88% relative to the free enzyme. The optimum crosslinking time was 30 min. The reusability of the immobilized enzyme was confirmed by the presence of residual activity after six cycles of reuse. Furthermore, the immobilized enzyme retains 40% of the initial activity after 14 days of storage. This work provides evidence that supports electrospinning as an effective and versatile technique for the encapsulation and immobilization of enzymes in polymer nanofibers. The known sensibility of papain toward metal ions as Hg2 + presents the possibility of its application in the fabrication of biosensors specialized in the detection of metal ions. Acknowledgments The funding support from CONACYT-Mexico (Project 174806, Convocatoria conjunta CONACYT-CNPq Mexico–Brasil en nanotecnologías 2011) and CONACYT (Project 176671, Convocatoria CienciaBásica 2012) as well as PROMEP-SEP-Mexico (103.5/12/4296)
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