Colloids and Surfaces B: Biointerfaces 128 (2015) 227–236
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Characterization and immobilization of trypsin on tannic acid modified Fe3 O4 nanoparticles Keziban Atacan a , Mahmut Özacar b,c,∗ a b c
Department of Chemistry, Graduate School of Natural and Applied Sciences, Sakarya University, Sakarya 54187, Turkey Department of Chemistry, Science & Arts Faculty, Sakarya University, Sakarya 54187, Turkey Biomedical, Magnetic and Semiconductor Materials Research Center (BIMAS-RC), Sakarya University, Sakarya 54187, Turkey
a r t i c l e
i n f o
Article history: Received 2 October 2014 Received in revised form 1 January 2015 Accepted 23 January 2015 Available online 3 February 2015 Keywords: Fe3 O4 nanoparticles Magnetic nanoparticles Trypsin Tannic acid Enzyme immobilization
a b s t r a c t Fe3 O4 nanoparticles (NPs) were synthesized by co-precipitating Fe2+ and Fe3+ in an ammonia solution. Fe3 O4 NPs functionalized with tannic acid were prepared. After functionalization process, trypsin enzyme was immobilized on these Fe3 O4 NPs. The influence of pH, temperature, thermal stability, storage time stability and reusability on non-covalent immobilization was studied. The properties of Fe3 O4 and its modified forms were examined by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), UV–vis spectrometer (UV) and X-ray diffraction (XRD), magnetization and zeta potential measurements. The immobilized enzyme was slightly more stable than the free enzyme at 45 ◦ C. According to the results, the activity of immobilized trypsin was preserved 55% at 45 ◦ C after 2 h and 90% after 120 days storage. In addition, the activity of the immobilized trypsin was preserved 40% of its initial activity after eight times of successive reuse. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanoparticles (NPs) are one of the most important building blocks in fabrication of nanomaterials. Their basic properties, extremely small size and high surface-area-to-volume ratio, provide a number of active centers for functionalization and enzyme immobilization. However, for such an application, it is necessary to use a method of purification that does not generate secondary waste and involves materials that can be recycled and easily used on an industrial scale [1]. Among the various nanostructures, magnetic nanoparticles (MNPs) have wide range of applications in the immobilization of cells and enzymes, bioseparation systems, immunoassays, drug delivery and biosensors [2–4]. The MNPs can easily be stabilized in a fluidized bed reactor for continuous operation of enzyme [4]. They are offered many attractive features in biotechnology [5] and possess unique properties, namely superparamagnetism, low toxicity and biocompatibility [6]. Functionalization of MNPs can be done by bonding different compounds which have the structure containing amino ( NH2 ),
∗ Corresponding author at: Department of Chemistry, Science & Arts Faculty, Sakarya University, Sakarya 54187, Turkey. Tel.: +90 264 295 60 41; fax: +90 264 295 59 50. E-mail address: [email protected] (M. Özacar). http://dx.doi.org/10.1016/j.colsurfb.2015.01.038 0927-7765/© 2015 Elsevier B.V. All rights reserved.
hydroxide ( OH), carboxylic acid ( COOH) or phosphate functional groups that allow immobilization of biomolecules like proteins, enzymes or antibodies. There are two main ways to immobilize biological particles: by physical adsorption and by covalent immobilization [7,8]. Immobilized enzymes usually show better thermal and pH stability, are easier to separate, can be reused and in effect appear to be more suitable for practical applications. As a result, immobilized biocatalysts have been utilized in a large number of practical applications such as high-tonnage processes in food and pharmaceutical industry or as bioseparators or biosensors [9,10]. Among the many methods of immobilization (e.g. adsorption, entrapment, electrostatic interaction and covalent binding) the covalent binding of enzymes to water-insoluble carriers seems to be the most attractive method for enzyme stabilization, reusability and recovery [11]. The immobilization of enzymes has been a booming field of research due to its advantages for the recovery and reuse of higher cost enzymes and for the easier separation of enzymes from catalytic product, as well as in the improvement of the enzymes’ stability in both storage and operational processes [12]. Trypsin (EC 3.4.21.4) is a member of a large family of serine proteinases, composed of a single polypeptide chain of 223 amino acid residues, and it acts by cleaving the ester and peptide bonds involving the carboxyl groups of arginine and lysine [13]. Generally, trypsin is isolated from the pancreas of mammals [14]. Trypsin has been commonly used in biological research during
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proteomics experiments. The growing demand for trypsin with special properties has driven attempts to chemically and physically modify it. Trypsin has been chemically modified by activated polyethylene glycol to improve its biological activity and thermal stability [15,16]. The immobilization of trypsin on both natural and synthetic carriers has been reported in many papers and carriers based on natural polysaccharides [17–20], mesoporous silicates, silica gels and glass [21–23] and synthetic polymers [24–28] have been studied progressively. Immobilization of the enzyme offers the advantages of enhanced stability, protection against autolysis and the possibility of repeated use of the catalytic material. However, the preparation of a successfully immobilized enzyme depends very much on the method of immobilization employed and on the chemical characteristics of the support. In particular the lateral chain groups of the enzyme that are implicated in binding to the support must be distant from the active site in order to avoid steric hindrance. This factor is particularly important for enzymes acting on voluminous substrates that require entering the active site to be hydrolyzed, and this factor may have a profound effect on the resulting biocatalytic activity [29]. The aim of this study was to characterize and immobilize the trypsin produced by bovine pancreas on Fe3 O4 nanoparticles coated with tannic acid. Trypsin was selected for enzyme immobilization due to its use in the numerous biotechnological processes. Trypsin was immobilized on the tannic acid modified Fe3 O4 nanoparticles. Also, the effect of temperature, pH and thermal/storage stability was also investigated. The processes of immobilization of trypsin via the tannic acid on Fe3 O4 nanoparticles are shown schematically in Fig. 1. 2. Experimental 2.1. Materials and methods Ferric chloride hexahydrate (FeCl3 ·6H2 O, >99%), ferrous sulfate heptahydrate (FeSO4 ·7H2 O, >99%), tris(hydroxymethyl)aminomethane (99.8–100.1%), ammonium hydroxide (25%, w/w), sodium hydroxide (≥97%), and ethanol (>99.2%) were obtained from Merck (Germany). Trypsin from bovine pancreas, N␣benzoyl-dl-arginine 4-nitroanilide hydrochloride (BAPNA) (98%), albumin from bovine serum (BSA) (98%, agarose gel electrophoresis), Bradford Reagent, and 4-nitroaniline (p-nitroaniline) were purchased from Sigma–Aldrich (USA). All aqueous solutions were prepared with deionized water that had been passed through a Millipore Milli-Q Plus water purification system. All chemicals were of analytical grade and used as received. 2.2. Synthesis of Fe3 O4 nanoparticles Fe3 O4 nanoparticles (MNPs) were prepared via improved chemical coprecipitation method [30]. According to this method, 4.4483 g of FeSO4 ·7H2 O (0.016 mol) and 7.5684 g of FeCl3 ·6H2 O (0.028 mol) were dissolved in 320 mL of deionized water, such that Fe2+ /Fe3+ = 1/1.75 [31]. The mixed solution was stirred at 200 rpm under N2 at 80 ◦ C for 1 h. Then, 40 mL of NH3 ·H2 O was injected into the mixture rapidly, stirred under N2 for another 1 h and then cooled to room temperature. The precipitated particles were washed five times with hot water and separated by magnetic decantation. Finally, MNPs were dried at 70 ◦ C in a vacuum oven overnight. 2.3. Fe3 O4 nanoparticles modified by tannic acid 1.0551 g Fe3 O4 NPs were sonicated in 40 mL water for 15 min to get uniform dispersion and mixed at 200 rpm under N2 atmosphere
at 40 ◦ C for 1 h. Then tannic acid solution (0.5 g/20 mL deionized water) was added to dispersion and mixed at 200 rpm under N2 atmosphere at 40 ◦ C for 2 h. After that the mixture was cooled to room temperature. The prepared tannic acid-modified Fe3 O4 NPs (TA-MNPs) were collected with a magnet, and washed with ethanol, followed with deionized water for three times. Finally, TA-MNPs were dried at 70 ◦ C in a vacuum oven overnight. 2.4. Immobilization of trypsin on tannic acid-modified Fe3 O4 NPs 0.100 g of TA-MNPs was put in an Erlenmeyer. Then, an appropriate amount of trypsin enzyme in sodium phosphate buffer solution (PBS) (0.1 M, pH 7.5) with a concentration of 1 mg/mL was added to tannic acid-modified Fe3 O4 NPs and the mixture was incubated by stirring at 200 rpm at 4 ◦ C for 3 h, then separated magnetically and washed with own buffer solution for three times. The prepared Fe3 O4 –tannic acid–trypsin (TTA-MNPs) was stored at 4 ◦ C until the using time. 2.5. Enzyme activity assay The enzymatic activity of free and immobilized trypsin was assayed using the chromogenic substrate N␣-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BAPNA), which gives yellow-colored p-nitroaniline (p-NA) upon hydrolysis and can be monitored at 410 nm by a Shimadzu UV-2401PC spectrophotometer. The enzymatic activity of free trypsin was measured by hydrolysis of 0.1% BAPNA solution (10 mg BAPNA, 0.2 mL DMSO, 9.8 mL deionized water) for 10 min at room temperature. The enzymatic activity of immobilized trypsin was measured by hydrolysis of 0.1% BAPNA for 10 min at room temperature and at 200 rpm in an orbital shaker. After incubation, immobilized trypsin was separated by magnetic decantation. One unit (U) of trypsin activity was expressed as the amount of enzyme that formed 1 mol of p-nitroaniline per minute under optimum reaction conditions [32,33]. 2.6. Protein assay Protein concentration of trypsin was determined according to the method of Bradford [34] using Bio-Rad protein dye reagent concentrate. Bovine serum albumin was used as the standard. The amount of bound protein was calculated from the difference between the amount of protein introduced into the coupling reaction mixture and the amount of protein present in the washing water after immobilization. 2.7. Characterization of support Thermogravimetric analysis (TGA) was carried out with Setaram thermogravimetric analyzer (Setsys Evolution, France). The sample weight was 15–17 mg. Analysis was performed from room temperature to 1000 ◦ C at a heating rate of 10 ◦ C/min in an argon atmosphere with a gas flow rate of 20 mL/min. The particle size and morphology of the samples were established by scanning electron microscopy (SEM), utilizing a JEOL JSM-6060 LV operated at 20 kV electron microscope. The atomic and molecular structure of crystalline Fe3 O4 NPs were investigated by X-ray diffraction (XRD, RIGAKU D max 2200 X-ray diffractometer with Cu KR () 0.154 nm radiation). FT-IR analysis was carried out using a Schimadzu IR Prestige 21 Fourier transformation infrared spectrometer. UV–vis spectra were obtained with a Shimadzu UV2401PC recording spectrophotometer. The magnetization curves of samples were measured with a vibrating sample magnetometry (VSM, LakeShore-7407, USA) at room temperature and zeta potential was measured at 30 ◦ C in Malvern Zeta Sizer (Nano-ZS).
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Fig. 1. Immobilization of trypsin on tannic acid modified Fe3 O4 nanoparticles.
2.8. Effect of pH and temperature on activity The effect of pH on activity of free and immobilized trypsin was assayed in the different buffers (0.1 M) of pH ranging from 4 to 10.5 by using the standard activity assay procedure mentioned above. The effect of temperature on free and immobilized trypsin was studied in the temperature range 4–85 ◦ C. Both forms of enzyme were incubated in sodium phosphate buffer (0.1 M, pH 7.5) for 30 min at different temperatures and, after cooling; the remaining activity was analyzed under the standard conditions. The relative activity (%) was calculated by the percentage of activity remaining compared to the initial activity at each time point for each sample. 2.9. Thermal and storage stability, reusability Thermal stabilities of both free and immobilized trypsin preparations were determined by measuring the residual activity of the enzyme exposed to 45 ◦ C in 0.1 M sodium phosphate buffer (pH 7.5) for different incubation times (30–120 min), respectively. Free and immobilized enzymes were stored at 4 ◦ C in 0.1 M sodium phosphate buffer (pH 7.5). The storage stability of enzymes was determined by measuring the activity of samples taken at regular time intervals and then compared. The residual activities were determined as above. In order to determine the reusability behavior of immobilized trypsin, the TTA-MNPs were stored at 4 ◦ C in 0.1 M PBS buffer (pH 7.5) for a period of 1 month. The reusability test of TTA-MNPs was performed at 30 ◦ C and it was the same as used for the enzyme activity assay in eight repeated cycles at every 4 days interval. For every repetitive cycle, the TTA-MNPs were removed from the reaction medium and washed with 0.1 M PBS buffer (pH 7.5) 5–6 times and stored in the same buffer for further use. 2.10. Statistical analysis All experimental measurements were carried out in triplicate. The results were expressed as the mean ± standard deviation (SD). The statistical significance of the differences in each experiment
was performed using one-way analysis of variance (ANOVA). Significance was accepted with P < 0.05. 3. Results and discussion 3.1. XRD studies of Fe3 O4 NPs The crystalline structure of pure Fe3 O4 NPs was measured by XRD method and the XRD pattern was shown in Fig. 2. The seven different characteristic peaks were marked to spot belonging to Fe3 O4 NPs 18.38, 30.24, 35.58, 43.34, 53.74, 57.3, and 62.8 two theta angles respectively to be (1 1 1), (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) [35,36]. All diffraction peaks in the pattern could be indexed as the spinel structure displayed by pure Fe3 O4 (JCPDS No. 82-1533), indicating that the samples exhibited a face centered cubic crystal system. No other reflection peaks than those from magnetite were detected, indicating the high purity and good crystallinity of exclusive Fe3 O4 products. 3.2. FTIR spectroscopy Fig. 3 depicts the FTIR spectra of the MNPs, TA-MNPs and TTAMNPs. The FTIR bands at low wave numbers (≤700 cm−1 ) were obtained from vibrations of Fe O bonds of iron oxide. The presence of MNPs can be seen by two strong absorption bands at around 632 and 585 cm−1 [37,38]. The spectrum (a) shows the only characteristic strong absorption bands at 561 cm−1 , 1396 cm−1 and 1630 cm−1 which come from vibrations of Fe O bonds and one typical for iron oxide [39]. The spectrum (b) is different from the spectrum (a) between 1000 cm−1 and 1700 cm−1 absorption bands. The C O group can be seen from the spectrum (b) at around 1706 cm−1 . The presence of the anchored tannic acid group was confirmed by O H or C H group stretching vibrations that appeared at 2925 and 2860 cm−1 . The broad peak in the region of 3600–3100 cm−1 is characteristic of the OH stretchings of the phenolic and methylol group of tannic acid [40–42]. The absorption bands between 1600
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(422) (222)
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Two-theta(deg) Fig. 2. XRD pattern of synthesized Fe3 O4 NPs.
and 1400 cm−1 are related to aromatic C C bonds. The peaks at 1343 and 1048 cm−1 in the spectrum of tannin belong to phenol groups [43,44]. When the spectra of TTA-MNPs were compared with TA-MNPs, around not only 1060 cm−1 but also the relative peak intensities of peak series at 1000 and 1700 cm−1 region changed due to proteintannate complex formation between trypsin enzyme and some phenolic groups of tannin.
The changes in the spectrum of TTA-MNPs after the immobilization reaction between trypsin enzyme and phenolic groups of TAMNPs are observed at both 1000–1700 cm−1 and 3600–3100 cm−1 regions for the spectrum of TTA-MNPs, because all of the OH groups in the tannin molecules do not participate in the complexation or covalent bond formation reactions between the tannin and trypsin enzyme. The phenolic groups participating immobilization or complexation reactions are located in the 1700–1000 cm−1
1124
1047,77
1396,2 1630,68
(a)
Transmission(%)
1069,3 3433,97 1343,4
1203,90
632 561,04
1706 1634,26
(b) 2860 2925
3435,60
1208
633,37 562,42 1060,86
1354 1513 1632,74
(c)
630
3443
4000
3500
3000
2500
2000
1500
Wavenumber(cm-1) Fig. 3. FTIR spectra of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs.
1000
559,21 500 450
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Fig. 4. SEM images of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs.
region for TA-MNPs. The wide bands in the 3600–3100 cm−1 region are not only participate reactions with trypsin enzyme but also belonging to free hydroxyl groups of the TA-MNPs [45,46]. 3.3. Scanning electron microscopy The co-precipitation process to obtain Fe3 O4 NPs was carried out in an alkaline aqueous medium and the final product obtained from this process yielded a dense, black and magnetic powder. SEM images shown in Fig. 4 reveal a heterogeneous morphology without porosity for MNPs, TA-MNPs and TTA-MNPs. However, these images are presented in a bigger mean size. These nanoparticles can be considered as microparticles with sizes approximately less than 5 m in Fig. 4. In fact, MNPs, TA-MNPs and TTA-MNPs are size less than 100 nm. But the SEM images cannot be obtained clearly in lower size. The modification of MNPs produced changes in the size particle and this can be attributed to the multi-core/shell structures [47,48]. 3.4. TGA The thermal stabilities of the MNPs, TA-MNPs and TTA-MNPs were evaluated by thermal gravimetric analysis (TGA) and its first derivative (dTGA) (Fig. 5(A) and (B)). Upon heating in TGA, MNPs show a weight loss of about 0.8% at temperatures ranging from 65 to 120 ◦ C, mainly due to the loss of physically adsorbed water on the material [31]. The weight loss of TA-MNPs was about 9.86% in a broad temperature range between 50 and 1000 ◦ C. Particularly, the weight loss of TA-MNPs was obtained about 7.8% between 150 and 450 ◦ C. The weight loss of TTA-MNPs was obtained about 11.35% in a broad temperature range between 50 and 1000 ◦ C. The decomposition of both TA-MNPs and TTA-MNPs systems essentially occurs in three stages. In the first stage, nearly 1.5% weight loss occurred between 50 and 200 ◦ C, which may be because of the loss of water molecules that had been bound with hydroxyl groups present in tannin and in the voids. In the second stage, the
decomposition temperature range of 200–450 ◦ C could be the result of the partial breakdown of the intermolecular bonding, stripping of the ring, chain cleavage, and elimination of volatile fractions. The weight loss of TA-MNPs and TTA-MNPs in this stage occurred as 7.5% and 8%, respectively. The third stage occurs in the temperature range of 450–700 ◦ C, which may be due to the oxidative degradation, and the tannic acid and trypsin molecules as a whole are decomposed with 9.86% and 11.35% total weight loss for TA-MNPs and TTA-MNPs, respectively. 3.5. Magnetic measurements The magnetic properties of the MNPs were measured at room temperature with VSM. Fig. 6 shows the hysteresis loops of the samples. The saturation magnetization was found to be 62.19 emu/g for TTA-MNPs, less than MNPs (69.51 emu/g) but a little bit more than TA-MNPs (60.98 emu/g). This difference suggests that a large amount of tannic acid coated on the surface of MNPs. As shown in the figure, no reduced remanence and coercivity being zero were detected, indicating that all MNPs, TA-MNPs and TTA-MNPs are superparamagnetic. When the external magnetic field was removed, the MNPs could be well dispersed by gentle shaking. These magnetic properties are critical in the applications of the biomedical and bioengineering fields. 3.6. Zeta potential Fig. 7 depicts the zeta-potential curves of aqueous dispersion of TA-MNPs and TTA-MNPs. The zeta potential value of TA-MNPs and TTA-MNPs gives high negative potential value varying pH ranges from 4.0 to 11.0. There were many literatures that reported the binding of protein and enzyme through electrostatic interaction with different support materials [49–51]. The isoelectric point of trypsin (pI) is 10.5. From zeta potential measurement, TA-MNPs contained highly surface negative charges due to OH group on tannic acid (zeta potential is −34.9 mV). The immobilization of
Table 1 Comparison of immobilization yield, optimum pH and temperature of immobilized trypsin in the present work and other studies. Support materials Fe3 O4 –TA PANIG Silica–GA Nochitosan modified microspheres (M1–M3) and chitosan modified microspheres (M4–M8) ChGly (activated with glutaraldehyde on chitosan gels) Amine-spent grain with glutaraldehyde PVAG–PANIG Trypsin from the digestive system of carp Catla catla
Immobilization yield (%) 81.1 – 63 – ≈100 61.3 – –
Optimum temperature (◦ C)
Optimum pH
References
45 45 45 40
9 7.6 9.5 8 and 8.5
This study [29] [33] [56]
55 50–60 35 40
7 7–9 7.6 7
[57] [58] [59] [64]
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658.2 81.4 238.1
312.9
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816 669.4 79.3
288.7
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800
Temperature (ºC) Fig. 5. TGA (A) and their first derivatives (B) of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs.
900
100 0
trypsin was carried in sodium phosphate buffer solution (0.1 M, pH 7.5). At this pH, trypsin was positively charged and TA-MNPs contained highly negative charges on its surface. As a result, the binding of trypsin on TA-MNPs took place through electrostatic interaction. Therefore, the interaction between trypsin and TA-MNPs was not the simple adsorption and the covalent immobilization, but non-covalent interactions may have occurred between nitrogen and sulfur atoms of enzyme and oxygen atoms of TA-MNPs [52]. The zeta potential values of TTA-MNPs were increased at the pH values below the isoelectric point of trypsin, indicating that the trypsin neutralized the negative charge of TA-MNPs during immobilization, but its zeta potential values were decreased above the isoelectric point of trypsin after the trypsin immobilization. It suggested that the immobilized trypsin did not neutralize the negative charge of TA-MNPs, which permitted the trypsin to site at the stern layer.
Magnetic Moment (emu/g)
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75 50
(a) (b)
25
(c)
0 -25 -50 -75 -10000
T= 300 K -5000
0
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Magnetic Field (Oe) Fig. 6. Magnetization curves of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs at 300 K.
3.7. Effect of pH and temperature on the activity of immobilized trypsin In order to determine the pH stabilities, free and immobilized enzymes were incubated at different pH values in 0.1 M buffer solutions at room temperature for 1 h in the range of pH 6.0–11.0 and the remaining activity was measured under standard activity assay conditions. pH is one of the most important factors influencing not only the properties of amino acid side groups but also the solution chemistry of the insoluble support. Thus, protein support interaction and surface properties of a protein are strongly influenced by the pH. While the optimum pH value of free trypsin was determined at pH 7.5, the immobilized trypsin was most active in the range of pH 8.5–9.5 in Fig. 8(A). These results were expected because the number of positively charged groups of enzyme linked with the amino groups to the carrier decreases after immobilization. Thus the character of the enzyme becomes more polyanionic [53]. In general, the optimum pH values of animal-origin trypsin are in the range of pH 6.0–9.0 [54,55]. These values were comparable to the optimum pH of some other support materials for trypsin (Table 1). For example, the optimum pH values of trypsin on Silica-GA and on chitosan modified microspheres were determined at pH 9.5 and 8.5, respectively [33,56]. In addition, the optimum pH values of trypsin on PANIG [29], ChGly [57], amine-spent grain with glutaraldehyde [58] and PVAG-PANIG [59] were found between pH 7 and 8. Although the activity of free trypsin readily decreased because of denaturation by a change in the pH value, the denaturation
of trypsin molecules would be considerably reduced by immobilization. It may be assumed that the trypsin molecule possesses terminal amino groups, and some amino groups of trypsin molecule would react with carboxyl groups of tannic acid on the surface of magnetic nanoparticles. The enhancement in the enzymatic activity of immobilized trypsin compared to free trypsin activity can be ascribed to both minimization/elimination of autolysis of trypsin and the possible stabilization of trypsin immobilized on magnetic nanoparticles with high surface area and confinement, resulting in higher accessibility of the substrate to the active sites of trypsin enzyme. This stabilization of trypsin activity is most probably the result of the multipoint linkages of trypsin and tannic acid molecules on the magnetic nanoparticle surface, which prevent not only the autolysis but also the trypsin denaturation [60,61]. The maximum activity was observed at 45 ◦ C for both forms of enzyme (Fig. 8(B)) and, generally, the optimum temperatures of animal-origin trypsin can typically be found in the range of 45–65 ◦ C [54,62,63]. The values of optimum temperature for this study and similar studies [29,33] was 45 ◦ C, whereas it was 40 ◦ C for other studies [56,64] (Table 1). At 50–85 ◦ C temperature range, the immobilized trypsin exhibited higher activity than the free enzyme, suggesting that it has a higher thermal stability. The increase in optimum temperature was caused by the changing physical and chemical properties of the trypsin. The prevention of thermal denaturation would also probably come from the multipoint
Fig. 7. Zeta potential curves for TA-MNPs and TTA-MNPs at various pH (error bars represent ±standard deviations, n = 3).
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(A)
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Temperature (°C) Fig. 8. Effect of pH (A) and temperature (B) on the activity of free and immobilized trypsin (error bars represent ±standard deviations, n = 3).
attachment between the trypsin molecules and the tannic acid molecules. The higher stability of the immobilized trypsin could be caused by the diminished autolysis, as the functional groups of enzyme molecules on MNPs have greatly restricted contact with each other [65–67]. 3.8. Thermal stability, storage stability and reusability on the activity of immobilized trypsin One of the most important parameters which affects the stability of enzymes is the temperature. Enzymes are usually more stable at lower temperatures while rapid thermal denaturation occurs at high temperatures. Time-dependent thermal stability of free and immobilized trypsin was determined at 45 ◦ C as shown in Fig. 9(A). The immobilized enzyme was found to be more thermostable than the free enzyme. Immobilized enzyme and free enzyme retained 55% and 45% of activity, respectively, at 45 ◦ C after 2 h. These results showed that the thermostability of the immobilized trypsin improved considerably as a result of immobilization. In several studies of bovine pancreatic trypsin, the enzyme preserves a large proportion of activity until 50 ◦ C, and then it rapidly loses activity above this temperature [68,69]. The high cost of enzymes used for industrial purposes and the time necessary for their immobilization have led to increasing interest in the storage stability of these enzymes for longer periods. Storage stability is an important advantage of immobilized enzymes over the free enzymes, because free enzymes may quickly
lose their activity. Both free and immobilized trypsin were stored at 4 ◦ C under the same conditions and the activity measurements were carried out for a 120-day period. Under the same storage conditions, the free enzyme was found to lose about 40% of its initial activity over this 120-day period, whereas the immobilized enzyme lost only about 10% of its initial activity over the same period of time (Fig. 9(B)). As can be seen from Fig. 9, the immobilized trypsin exhibits higher storage stability than that of the free form. The generated multipoint covalent interaction between enzyme and the carboxyl groups of the tannic acid on the MNPs surface should also convey a higher conformational stability to the immobilized trypsin. The relative severe decrease in activity of the free trypsin might be due to its susceptible autolysis during the storage time. It should be noted that the immobilization of trypsin on the TA-MNPs could decrease the autolysis effect [67]. The most important goals of enzyme immobilization are the recycling and the reuse of the enzyme after it is used in the process. The reusability of the immobilized enzyme in sodium phosphate buffer solution (0.1 M, pH 7.5) was investigated for this purpose. Activity of the immobilized trypsin was measured eight times over a period of 1 month as shown in Fig. 9(C). For every repetitive cycle, the immobilized trypsin was washed with sodium phosphate buffer solution (0.1 M, pH 7.5) and easily separated from product by magnet and then stored in the same buffer for further use. The immobilized trypsin was retained 40% of its initial activity after eight times of successive reuse. The immobilized enzymes are reused many times, different from the free enzyme, which is used only once.
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(A)
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120
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Cycle Number Fig. 9. Effect of thermal (A) and storage stability (B) on the activity of free and immobilized trypsin and (C) reusability of immobilized trypsin (error bars represent ±standard deviations, n = 3).
4. Conclusions Trypsin was immobilized on tannic acid-modified Fe3 O4 NPs by using covalent binding method. The immobilized enzyme with good stabilities and reusability increases the potential use of trypsin for different biotechnological applications. So, we thought that tannic acid-modified Fe3 O4 NPs were a kind of good precursor for the immobilization of trypsin and were
worthy of widespread applications because tannic acid is a natural, harmless and abundant polyphenol. All these results demonstrate that the tannic acid modified magnetic nanoparticles are good supports for enzyme immobilization and will find more biological applications including enzyme engineering, drug delivery, biofuel cell, etc. Thus, the presented TA-MNPs can provide economic advantages for large scale biotechnological applications.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Characterization and immobilization of trypsin on tannic acid modified Fe3 O4 nanoparticles Keziban Atacan a , Mahmut Özacar b,c,∗ a b c
Department of Chemistry, Graduate School of Natural and Applied Sciences, Sakarya University, Sakarya 54187, Turkey Department of Chemistry, Science & Arts Faculty, Sakarya University, Sakarya 54187, Turkey Biomedical, Magnetic and Semiconductor Materials Research Center (BIMAS-RC), Sakarya University, Sakarya 54187, Turkey
a r t i c l e
i n f o
Article history: Received 2 October 2014 Received in revised form 1 January 2015 Accepted 23 January 2015 Available online 3 February 2015 Keywords: Fe3 O4 nanoparticles Magnetic nanoparticles Trypsin Tannic acid Enzyme immobilization
a b s t r a c t Fe3 O4 nanoparticles (NPs) were synthesized by co-precipitating Fe2+ and Fe3+ in an ammonia solution. Fe3 O4 NPs functionalized with tannic acid were prepared. After functionalization process, trypsin enzyme was immobilized on these Fe3 O4 NPs. The influence of pH, temperature, thermal stability, storage time stability and reusability on non-covalent immobilization was studied. The properties of Fe3 O4 and its modified forms were examined by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), UV–vis spectrometer (UV) and X-ray diffraction (XRD), magnetization and zeta potential measurements. The immobilized enzyme was slightly more stable than the free enzyme at 45 ◦ C. According to the results, the activity of immobilized trypsin was preserved 55% at 45 ◦ C after 2 h and 90% after 120 days storage. In addition, the activity of the immobilized trypsin was preserved 40% of its initial activity after eight times of successive reuse. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nanoparticles (NPs) are one of the most important building blocks in fabrication of nanomaterials. Their basic properties, extremely small size and high surface-area-to-volume ratio, provide a number of active centers for functionalization and enzyme immobilization. However, for such an application, it is necessary to use a method of purification that does not generate secondary waste and involves materials that can be recycled and easily used on an industrial scale [1]. Among the various nanostructures, magnetic nanoparticles (MNPs) have wide range of applications in the immobilization of cells and enzymes, bioseparation systems, immunoassays, drug delivery and biosensors [2–4]. The MNPs can easily be stabilized in a fluidized bed reactor for continuous operation of enzyme [4]. They are offered many attractive features in biotechnology [5] and possess unique properties, namely superparamagnetism, low toxicity and biocompatibility [6]. Functionalization of MNPs can be done by bonding different compounds which have the structure containing amino ( NH2 ),
∗ Corresponding author at: Department of Chemistry, Science & Arts Faculty, Sakarya University, Sakarya 54187, Turkey. Tel.: +90 264 295 60 41; fax: +90 264 295 59 50. E-mail address: [email protected] (M. Özacar). http://dx.doi.org/10.1016/j.colsurfb.2015.01.038 0927-7765/© 2015 Elsevier B.V. All rights reserved.
hydroxide ( OH), carboxylic acid ( COOH) or phosphate functional groups that allow immobilization of biomolecules like proteins, enzymes or antibodies. There are two main ways to immobilize biological particles: by physical adsorption and by covalent immobilization [7,8]. Immobilized enzymes usually show better thermal and pH stability, are easier to separate, can be reused and in effect appear to be more suitable for practical applications. As a result, immobilized biocatalysts have been utilized in a large number of practical applications such as high-tonnage processes in food and pharmaceutical industry or as bioseparators or biosensors [9,10]. Among the many methods of immobilization (e.g. adsorption, entrapment, electrostatic interaction and covalent binding) the covalent binding of enzymes to water-insoluble carriers seems to be the most attractive method for enzyme stabilization, reusability and recovery [11]. The immobilization of enzymes has been a booming field of research due to its advantages for the recovery and reuse of higher cost enzymes and for the easier separation of enzymes from catalytic product, as well as in the improvement of the enzymes’ stability in both storage and operational processes [12]. Trypsin (EC 3.4.21.4) is a member of a large family of serine proteinases, composed of a single polypeptide chain of 223 amino acid residues, and it acts by cleaving the ester and peptide bonds involving the carboxyl groups of arginine and lysine [13]. Generally, trypsin is isolated from the pancreas of mammals [14]. Trypsin has been commonly used in biological research during
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proteomics experiments. The growing demand for trypsin with special properties has driven attempts to chemically and physically modify it. Trypsin has been chemically modified by activated polyethylene glycol to improve its biological activity and thermal stability [15,16]. The immobilization of trypsin on both natural and synthetic carriers has been reported in many papers and carriers based on natural polysaccharides [17–20], mesoporous silicates, silica gels and glass [21–23] and synthetic polymers [24–28] have been studied progressively. Immobilization of the enzyme offers the advantages of enhanced stability, protection against autolysis and the possibility of repeated use of the catalytic material. However, the preparation of a successfully immobilized enzyme depends very much on the method of immobilization employed and on the chemical characteristics of the support. In particular the lateral chain groups of the enzyme that are implicated in binding to the support must be distant from the active site in order to avoid steric hindrance. This factor is particularly important for enzymes acting on voluminous substrates that require entering the active site to be hydrolyzed, and this factor may have a profound effect on the resulting biocatalytic activity [29]. The aim of this study was to characterize and immobilize the trypsin produced by bovine pancreas on Fe3 O4 nanoparticles coated with tannic acid. Trypsin was selected for enzyme immobilization due to its use in the numerous biotechnological processes. Trypsin was immobilized on the tannic acid modified Fe3 O4 nanoparticles. Also, the effect of temperature, pH and thermal/storage stability was also investigated. The processes of immobilization of trypsin via the tannic acid on Fe3 O4 nanoparticles are shown schematically in Fig. 1. 2. Experimental 2.1. Materials and methods Ferric chloride hexahydrate (FeCl3 ·6H2 O, >99%), ferrous sulfate heptahydrate (FeSO4 ·7H2 O, >99%), tris(hydroxymethyl)aminomethane (99.8–100.1%), ammonium hydroxide (25%, w/w), sodium hydroxide (≥97%), and ethanol (>99.2%) were obtained from Merck (Germany). Trypsin from bovine pancreas, N␣benzoyl-dl-arginine 4-nitroanilide hydrochloride (BAPNA) (98%), albumin from bovine serum (BSA) (98%, agarose gel electrophoresis), Bradford Reagent, and 4-nitroaniline (p-nitroaniline) were purchased from Sigma–Aldrich (USA). All aqueous solutions were prepared with deionized water that had been passed through a Millipore Milli-Q Plus water purification system. All chemicals were of analytical grade and used as received. 2.2. Synthesis of Fe3 O4 nanoparticles Fe3 O4 nanoparticles (MNPs) were prepared via improved chemical coprecipitation method [30]. According to this method, 4.4483 g of FeSO4 ·7H2 O (0.016 mol) and 7.5684 g of FeCl3 ·6H2 O (0.028 mol) were dissolved in 320 mL of deionized water, such that Fe2+ /Fe3+ = 1/1.75 [31]. The mixed solution was stirred at 200 rpm under N2 at 80 ◦ C for 1 h. Then, 40 mL of NH3 ·H2 O was injected into the mixture rapidly, stirred under N2 for another 1 h and then cooled to room temperature. The precipitated particles were washed five times with hot water and separated by magnetic decantation. Finally, MNPs were dried at 70 ◦ C in a vacuum oven overnight. 2.3. Fe3 O4 nanoparticles modified by tannic acid 1.0551 g Fe3 O4 NPs were sonicated in 40 mL water for 15 min to get uniform dispersion and mixed at 200 rpm under N2 atmosphere
at 40 ◦ C for 1 h. Then tannic acid solution (0.5 g/20 mL deionized water) was added to dispersion and mixed at 200 rpm under N2 atmosphere at 40 ◦ C for 2 h. After that the mixture was cooled to room temperature. The prepared tannic acid-modified Fe3 O4 NPs (TA-MNPs) were collected with a magnet, and washed with ethanol, followed with deionized water for three times. Finally, TA-MNPs were dried at 70 ◦ C in a vacuum oven overnight. 2.4. Immobilization of trypsin on tannic acid-modified Fe3 O4 NPs 0.100 g of TA-MNPs was put in an Erlenmeyer. Then, an appropriate amount of trypsin enzyme in sodium phosphate buffer solution (PBS) (0.1 M, pH 7.5) with a concentration of 1 mg/mL was added to tannic acid-modified Fe3 O4 NPs and the mixture was incubated by stirring at 200 rpm at 4 ◦ C for 3 h, then separated magnetically and washed with own buffer solution for three times. The prepared Fe3 O4 –tannic acid–trypsin (TTA-MNPs) was stored at 4 ◦ C until the using time. 2.5. Enzyme activity assay The enzymatic activity of free and immobilized trypsin was assayed using the chromogenic substrate N␣-benzoyl-dl-arginine 4-nitroanilide hydrochloride (BAPNA), which gives yellow-colored p-nitroaniline (p-NA) upon hydrolysis and can be monitored at 410 nm by a Shimadzu UV-2401PC spectrophotometer. The enzymatic activity of free trypsin was measured by hydrolysis of 0.1% BAPNA solution (10 mg BAPNA, 0.2 mL DMSO, 9.8 mL deionized water) for 10 min at room temperature. The enzymatic activity of immobilized trypsin was measured by hydrolysis of 0.1% BAPNA for 10 min at room temperature and at 200 rpm in an orbital shaker. After incubation, immobilized trypsin was separated by magnetic decantation. One unit (U) of trypsin activity was expressed as the amount of enzyme that formed 1 mol of p-nitroaniline per minute under optimum reaction conditions [32,33]. 2.6. Protein assay Protein concentration of trypsin was determined according to the method of Bradford [34] using Bio-Rad protein dye reagent concentrate. Bovine serum albumin was used as the standard. The amount of bound protein was calculated from the difference between the amount of protein introduced into the coupling reaction mixture and the amount of protein present in the washing water after immobilization. 2.7. Characterization of support Thermogravimetric analysis (TGA) was carried out with Setaram thermogravimetric analyzer (Setsys Evolution, France). The sample weight was 15–17 mg. Analysis was performed from room temperature to 1000 ◦ C at a heating rate of 10 ◦ C/min in an argon atmosphere with a gas flow rate of 20 mL/min. The particle size and morphology of the samples were established by scanning electron microscopy (SEM), utilizing a JEOL JSM-6060 LV operated at 20 kV electron microscope. The atomic and molecular structure of crystalline Fe3 O4 NPs were investigated by X-ray diffraction (XRD, RIGAKU D max 2200 X-ray diffractometer with Cu KR () 0.154 nm radiation). FT-IR analysis was carried out using a Schimadzu IR Prestige 21 Fourier transformation infrared spectrometer. UV–vis spectra were obtained with a Shimadzu UV2401PC recording spectrophotometer. The magnetization curves of samples were measured with a vibrating sample magnetometry (VSM, LakeShore-7407, USA) at room temperature and zeta potential was measured at 30 ◦ C in Malvern Zeta Sizer (Nano-ZS).
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Fig. 1. Immobilization of trypsin on tannic acid modified Fe3 O4 nanoparticles.
2.8. Effect of pH and temperature on activity The effect of pH on activity of free and immobilized trypsin was assayed in the different buffers (0.1 M) of pH ranging from 4 to 10.5 by using the standard activity assay procedure mentioned above. The effect of temperature on free and immobilized trypsin was studied in the temperature range 4–85 ◦ C. Both forms of enzyme were incubated in sodium phosphate buffer (0.1 M, pH 7.5) for 30 min at different temperatures and, after cooling; the remaining activity was analyzed under the standard conditions. The relative activity (%) was calculated by the percentage of activity remaining compared to the initial activity at each time point for each sample. 2.9. Thermal and storage stability, reusability Thermal stabilities of both free and immobilized trypsin preparations were determined by measuring the residual activity of the enzyme exposed to 45 ◦ C in 0.1 M sodium phosphate buffer (pH 7.5) for different incubation times (30–120 min), respectively. Free and immobilized enzymes were stored at 4 ◦ C in 0.1 M sodium phosphate buffer (pH 7.5). The storage stability of enzymes was determined by measuring the activity of samples taken at regular time intervals and then compared. The residual activities were determined as above. In order to determine the reusability behavior of immobilized trypsin, the TTA-MNPs were stored at 4 ◦ C in 0.1 M PBS buffer (pH 7.5) for a period of 1 month. The reusability test of TTA-MNPs was performed at 30 ◦ C and it was the same as used for the enzyme activity assay in eight repeated cycles at every 4 days interval. For every repetitive cycle, the TTA-MNPs were removed from the reaction medium and washed with 0.1 M PBS buffer (pH 7.5) 5–6 times and stored in the same buffer for further use. 2.10. Statistical analysis All experimental measurements were carried out in triplicate. The results were expressed as the mean ± standard deviation (SD). The statistical significance of the differences in each experiment
was performed using one-way analysis of variance (ANOVA). Significance was accepted with P < 0.05. 3. Results and discussion 3.1. XRD studies of Fe3 O4 NPs The crystalline structure of pure Fe3 O4 NPs was measured by XRD method and the XRD pattern was shown in Fig. 2. The seven different characteristic peaks were marked to spot belonging to Fe3 O4 NPs 18.38, 30.24, 35.58, 43.34, 53.74, 57.3, and 62.8 two theta angles respectively to be (1 1 1), (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) [35,36]. All diffraction peaks in the pattern could be indexed as the spinel structure displayed by pure Fe3 O4 (JCPDS No. 82-1533), indicating that the samples exhibited a face centered cubic crystal system. No other reflection peaks than those from magnetite were detected, indicating the high purity and good crystallinity of exclusive Fe3 O4 products. 3.2. FTIR spectroscopy Fig. 3 depicts the FTIR spectra of the MNPs, TA-MNPs and TTAMNPs. The FTIR bands at low wave numbers (≤700 cm−1 ) were obtained from vibrations of Fe O bonds of iron oxide. The presence of MNPs can be seen by two strong absorption bands at around 632 and 585 cm−1 [37,38]. The spectrum (a) shows the only characteristic strong absorption bands at 561 cm−1 , 1396 cm−1 and 1630 cm−1 which come from vibrations of Fe O bonds and one typical for iron oxide [39]. The spectrum (b) is different from the spectrum (a) between 1000 cm−1 and 1700 cm−1 absorption bands. The C O group can be seen from the spectrum (b) at around 1706 cm−1 . The presence of the anchored tannic acid group was confirmed by O H or C H group stretching vibrations that appeared at 2925 and 2860 cm−1 . The broad peak in the region of 3600–3100 cm−1 is characteristic of the OH stretchings of the phenolic and methylol group of tannic acid [40–42]. The absorption bands between 1600
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600 (311)
Intensity(Counts)
500
400 300 (440)
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100
(422) (222)
(111)
0 10
0
20
30
40
50
60
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80
90
100
Two-theta(deg) Fig. 2. XRD pattern of synthesized Fe3 O4 NPs.
and 1400 cm−1 are related to aromatic C C bonds. The peaks at 1343 and 1048 cm−1 in the spectrum of tannin belong to phenol groups [43,44]. When the spectra of TTA-MNPs were compared with TA-MNPs, around not only 1060 cm−1 but also the relative peak intensities of peak series at 1000 and 1700 cm−1 region changed due to proteintannate complex formation between trypsin enzyme and some phenolic groups of tannin.
The changes in the spectrum of TTA-MNPs after the immobilization reaction between trypsin enzyme and phenolic groups of TAMNPs are observed at both 1000–1700 cm−1 and 3600–3100 cm−1 regions for the spectrum of TTA-MNPs, because all of the OH groups in the tannin molecules do not participate in the complexation or covalent bond formation reactions between the tannin and trypsin enzyme. The phenolic groups participating immobilization or complexation reactions are located in the 1700–1000 cm−1
1124
1047,77
1396,2 1630,68
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Transmission(%)
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1203,90
632 561,04
1706 1634,26
(b) 2860 2925
3435,60
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633,37 562,42 1060,86
1354 1513 1632,74
(c)
630
3443
4000
3500
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Wavenumber(cm-1) Fig. 3. FTIR spectra of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs.
1000
559,21 500 450
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Fig. 4. SEM images of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs.
region for TA-MNPs. The wide bands in the 3600–3100 cm−1 region are not only participate reactions with trypsin enzyme but also belonging to free hydroxyl groups of the TA-MNPs [45,46]. 3.3. Scanning electron microscopy The co-precipitation process to obtain Fe3 O4 NPs was carried out in an alkaline aqueous medium and the final product obtained from this process yielded a dense, black and magnetic powder. SEM images shown in Fig. 4 reveal a heterogeneous morphology without porosity for MNPs, TA-MNPs and TTA-MNPs. However, these images are presented in a bigger mean size. These nanoparticles can be considered as microparticles with sizes approximately less than 5 m in Fig. 4. In fact, MNPs, TA-MNPs and TTA-MNPs are size less than 100 nm. But the SEM images cannot be obtained clearly in lower size. The modification of MNPs produced changes in the size particle and this can be attributed to the multi-core/shell structures [47,48]. 3.4. TGA The thermal stabilities of the MNPs, TA-MNPs and TTA-MNPs were evaluated by thermal gravimetric analysis (TGA) and its first derivative (dTGA) (Fig. 5(A) and (B)). Upon heating in TGA, MNPs show a weight loss of about 0.8% at temperatures ranging from 65 to 120 ◦ C, mainly due to the loss of physically adsorbed water on the material [31]. The weight loss of TA-MNPs was about 9.86% in a broad temperature range between 50 and 1000 ◦ C. Particularly, the weight loss of TA-MNPs was obtained about 7.8% between 150 and 450 ◦ C. The weight loss of TTA-MNPs was obtained about 11.35% in a broad temperature range between 50 and 1000 ◦ C. The decomposition of both TA-MNPs and TTA-MNPs systems essentially occurs in three stages. In the first stage, nearly 1.5% weight loss occurred between 50 and 200 ◦ C, which may be because of the loss of water molecules that had been bound with hydroxyl groups present in tannin and in the voids. In the second stage, the
decomposition temperature range of 200–450 ◦ C could be the result of the partial breakdown of the intermolecular bonding, stripping of the ring, chain cleavage, and elimination of volatile fractions. The weight loss of TA-MNPs and TTA-MNPs in this stage occurred as 7.5% and 8%, respectively. The third stage occurs in the temperature range of 450–700 ◦ C, which may be due to the oxidative degradation, and the tannic acid and trypsin molecules as a whole are decomposed with 9.86% and 11.35% total weight loss for TA-MNPs and TTA-MNPs, respectively. 3.5. Magnetic measurements The magnetic properties of the MNPs were measured at room temperature with VSM. Fig. 6 shows the hysteresis loops of the samples. The saturation magnetization was found to be 62.19 emu/g for TTA-MNPs, less than MNPs (69.51 emu/g) but a little bit more than TA-MNPs (60.98 emu/g). This difference suggests that a large amount of tannic acid coated on the surface of MNPs. As shown in the figure, no reduced remanence and coercivity being zero were detected, indicating that all MNPs, TA-MNPs and TTA-MNPs are superparamagnetic. When the external magnetic field was removed, the MNPs could be well dispersed by gentle shaking. These magnetic properties are critical in the applications of the biomedical and bioengineering fields. 3.6. Zeta potential Fig. 7 depicts the zeta-potential curves of aqueous dispersion of TA-MNPs and TTA-MNPs. The zeta potential value of TA-MNPs and TTA-MNPs gives high negative potential value varying pH ranges from 4.0 to 11.0. There were many literatures that reported the binding of protein and enzyme through electrostatic interaction with different support materials [49–51]. The isoelectric point of trypsin (pI) is 10.5. From zeta potential measurement, TA-MNPs contained highly surface negative charges due to OH group on tannic acid (zeta potential is −34.9 mV). The immobilization of
Table 1 Comparison of immobilization yield, optimum pH and temperature of immobilized trypsin in the present work and other studies. Support materials Fe3 O4 –TA PANIG Silica–GA Nochitosan modified microspheres (M1–M3) and chitosan modified microspheres (M4–M8) ChGly (activated with glutaraldehyde on chitosan gels) Amine-spent grain with glutaraldehyde PVAG–PANIG Trypsin from the digestive system of carp Catla catla
Immobilization yield (%) 81.1 – 63 – ≈100 61.3 – –
Optimum temperature (◦ C)
Optimum pH
References
45 45 45 40
9 7.6 9.5 8 and 8.5
This study [29] [33] [56]
55 50–60 35 40
7 7–9 7.6 7
[57] [58] [59] [64]
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(A)
TG/%
0
(a)
TGA (%)
-2
-4
-6
-8
(b)
-10
(c)
100
200
300
400
500
600
700
800
900
1000
Sıcaklık(ºC)
(B)
(a)
dTGA
246.2
64.4
(b)
836.3
658.2 81.4 238.1
312.9
(c)
816 669.4 79.3
288.7
100
200
300
400
500
600
700
800
Temperature (ºC) Fig. 5. TGA (A) and their first derivatives (B) of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs.
900
100 0
trypsin was carried in sodium phosphate buffer solution (0.1 M, pH 7.5). At this pH, trypsin was positively charged and TA-MNPs contained highly negative charges on its surface. As a result, the binding of trypsin on TA-MNPs took place through electrostatic interaction. Therefore, the interaction between trypsin and TA-MNPs was not the simple adsorption and the covalent immobilization, but non-covalent interactions may have occurred between nitrogen and sulfur atoms of enzyme and oxygen atoms of TA-MNPs [52]. The zeta potential values of TTA-MNPs were increased at the pH values below the isoelectric point of trypsin, indicating that the trypsin neutralized the negative charge of TA-MNPs during immobilization, but its zeta potential values were decreased above the isoelectric point of trypsin after the trypsin immobilization. It suggested that the immobilized trypsin did not neutralize the negative charge of TA-MNPs, which permitted the trypsin to site at the stern layer.
Magnetic Moment (emu/g)
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233
75 50
(a) (b)
25
(c)
0 -25 -50 -75 -10000
T= 300 K -5000
0
5000
10000
Magnetic Field (Oe) Fig. 6. Magnetization curves of (a) MNPs, (b) TA-MNPs and (c) TTA-MNPs at 300 K.
3.7. Effect of pH and temperature on the activity of immobilized trypsin In order to determine the pH stabilities, free and immobilized enzymes were incubated at different pH values in 0.1 M buffer solutions at room temperature for 1 h in the range of pH 6.0–11.0 and the remaining activity was measured under standard activity assay conditions. pH is one of the most important factors influencing not only the properties of amino acid side groups but also the solution chemistry of the insoluble support. Thus, protein support interaction and surface properties of a protein are strongly influenced by the pH. While the optimum pH value of free trypsin was determined at pH 7.5, the immobilized trypsin was most active in the range of pH 8.5–9.5 in Fig. 8(A). These results were expected because the number of positively charged groups of enzyme linked with the amino groups to the carrier decreases after immobilization. Thus the character of the enzyme becomes more polyanionic [53]. In general, the optimum pH values of animal-origin trypsin are in the range of pH 6.0–9.0 [54,55]. These values were comparable to the optimum pH of some other support materials for trypsin (Table 1). For example, the optimum pH values of trypsin on Silica-GA and on chitosan modified microspheres were determined at pH 9.5 and 8.5, respectively [33,56]. In addition, the optimum pH values of trypsin on PANIG [29], ChGly [57], amine-spent grain with glutaraldehyde [58] and PVAG-PANIG [59] were found between pH 7 and 8. Although the activity of free trypsin readily decreased because of denaturation by a change in the pH value, the denaturation
of trypsin molecules would be considerably reduced by immobilization. It may be assumed that the trypsin molecule possesses terminal amino groups, and some amino groups of trypsin molecule would react with carboxyl groups of tannic acid on the surface of magnetic nanoparticles. The enhancement in the enzymatic activity of immobilized trypsin compared to free trypsin activity can be ascribed to both minimization/elimination of autolysis of trypsin and the possible stabilization of trypsin immobilized on magnetic nanoparticles with high surface area and confinement, resulting in higher accessibility of the substrate to the active sites of trypsin enzyme. This stabilization of trypsin activity is most probably the result of the multipoint linkages of trypsin and tannic acid molecules on the magnetic nanoparticle surface, which prevent not only the autolysis but also the trypsin denaturation [60,61]. The maximum activity was observed at 45 ◦ C for both forms of enzyme (Fig. 8(B)) and, generally, the optimum temperatures of animal-origin trypsin can typically be found in the range of 45–65 ◦ C [54,62,63]. The values of optimum temperature for this study and similar studies [29,33] was 45 ◦ C, whereas it was 40 ◦ C for other studies [56,64] (Table 1). At 50–85 ◦ C temperature range, the immobilized trypsin exhibited higher activity than the free enzyme, suggesting that it has a higher thermal stability. The increase in optimum temperature was caused by the changing physical and chemical properties of the trypsin. The prevention of thermal denaturation would also probably come from the multipoint
Fig. 7. Zeta potential curves for TA-MNPs and TTA-MNPs at various pH (error bars represent ±standard deviations, n = 3).
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(A)
immobilized trypsin free trypsin
Relative Activity (%)
120 100 80 60 40 20 0 5,5
6
6,5
7
7,5
8
8,5
9
9,5
10
10,5
11
pH
(B)
free trypsin
Relative Activity (%)
120
immobilized trypsin
100 80 60 40 20 0 0
10
20
30
40
50
60
70
80
90
Temperature (°C) Fig. 8. Effect of pH (A) and temperature (B) on the activity of free and immobilized trypsin (error bars represent ±standard deviations, n = 3).
attachment between the trypsin molecules and the tannic acid molecules. The higher stability of the immobilized trypsin could be caused by the diminished autolysis, as the functional groups of enzyme molecules on MNPs have greatly restricted contact with each other [65–67]. 3.8. Thermal stability, storage stability and reusability on the activity of immobilized trypsin One of the most important parameters which affects the stability of enzymes is the temperature. Enzymes are usually more stable at lower temperatures while rapid thermal denaturation occurs at high temperatures. Time-dependent thermal stability of free and immobilized trypsin was determined at 45 ◦ C as shown in Fig. 9(A). The immobilized enzyme was found to be more thermostable than the free enzyme. Immobilized enzyme and free enzyme retained 55% and 45% of activity, respectively, at 45 ◦ C after 2 h. These results showed that the thermostability of the immobilized trypsin improved considerably as a result of immobilization. In several studies of bovine pancreatic trypsin, the enzyme preserves a large proportion of activity until 50 ◦ C, and then it rapidly loses activity above this temperature [68,69]. The high cost of enzymes used for industrial purposes and the time necessary for their immobilization have led to increasing interest in the storage stability of these enzymes for longer periods. Storage stability is an important advantage of immobilized enzymes over the free enzymes, because free enzymes may quickly
lose their activity. Both free and immobilized trypsin were stored at 4 ◦ C under the same conditions and the activity measurements were carried out for a 120-day period. Under the same storage conditions, the free enzyme was found to lose about 40% of its initial activity over this 120-day period, whereas the immobilized enzyme lost only about 10% of its initial activity over the same period of time (Fig. 9(B)). As can be seen from Fig. 9, the immobilized trypsin exhibits higher storage stability than that of the free form. The generated multipoint covalent interaction between enzyme and the carboxyl groups of the tannic acid on the MNPs surface should also convey a higher conformational stability to the immobilized trypsin. The relative severe decrease in activity of the free trypsin might be due to its susceptible autolysis during the storage time. It should be noted that the immobilization of trypsin on the TA-MNPs could decrease the autolysis effect [67]. The most important goals of enzyme immobilization are the recycling and the reuse of the enzyme after it is used in the process. The reusability of the immobilized enzyme in sodium phosphate buffer solution (0.1 M, pH 7.5) was investigated for this purpose. Activity of the immobilized trypsin was measured eight times over a period of 1 month as shown in Fig. 9(C). For every repetitive cycle, the immobilized trypsin was washed with sodium phosphate buffer solution (0.1 M, pH 7.5) and easily separated from product by magnet and then stored in the same buffer for further use. The immobilized trypsin was retained 40% of its initial activity after eight times of successive reuse. The immobilized enzymes are reused many times, different from the free enzyme, which is used only once.
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(A)
free trypsin
120
Relative Activity (%)
235
immobilized trypsin
100 80 60 40 20 0 0
30
60
90
120
Time (min)
(B)
free trypsin
Relative Activity (%)
120
immobilized trypsin
100 80 60 40 20 0 20
0
40
60
80
100
120
140
Storage Time (day)
İmmobilized trypsin
(C)
Relative Activity (%)
120 100 80 60 40 20 0 0
1
2
3
4
5
6
7
8
9
Cycle Number Fig. 9. Effect of thermal (A) and storage stability (B) on the activity of free and immobilized trypsin and (C) reusability of immobilized trypsin (error bars represent ±standard deviations, n = 3).
4. Conclusions Trypsin was immobilized on tannic acid-modified Fe3 O4 NPs by using covalent binding method. The immobilized enzyme with good stabilities and reusability increases the potential use of trypsin for different biotechnological applications. So, we thought that tannic acid-modified Fe3 O4 NPs were a kind of good precursor for the immobilization of trypsin and were
worthy of widespread applications because tannic acid is a natural, harmless and abundant polyphenol. All these results demonstrate that the tannic acid modified magnetic nanoparticles are good supports for enzyme immobilization and will find more biological applications including enzyme engineering, drug delivery, biofuel cell, etc. Thus, the presented TA-MNPs can provide economic advantages for large scale biotechnological applications.
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