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Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles
Accepted Manuscript Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles Junchen Feng, Siran Yu, Jian Li, Ting Mo, Ping Li PII: DOI: Reference:
S1385-8947(15)01494-1 http://dx.doi.org/10.1016/j.cej.2015.10.083 CEJ 14359
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
14 April 2015 10 October 2015 26 October 2015
Please cite this article as: J. Feng, S. Yu, J. Li, T. Mo, P. Li, Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles, Chemical Engineering Journal (2015), doi: http://dx.doi.org/10.1016/j.cej.2015.10.083
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Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles
Junchen Feng a, Siran Yu a, Jian Li b, Ting Mo a, Ping Li a,* a
School of Life Sciences and Technology, Tongji University, No. 1239 Siping Road, Shanghai 200092, China
b
School of Engineering, Anhui Agricultural University, No. 130 West Changjiang Road, Hefei 230036, China
* Corresponding author: Prof. Ping Li Tel.: +86 21 65981051 Fax: +86 21 65981041 E-mail address: [email protected]
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Abstract: Aminoacylase (EC 3.5.1.14) was immobilized via covalent bonding to magnetic iron oxide (Fe3O4) nanoparticles with 3-ammonia propyl triethoxy silane (APTES) modification. Magnetic Fe3O4 nanoparticles were prepared by chemical co-precipitation, controlling n(Fe3+):n(Fe2+)=2:1 and c(Fe3+)+c(Fe2+)=0.3 mol/L. The nanoparticles modified with APTES were characterized by SEM and FT-IR. The results showed that the optimal nanoparticle concentration, glutaraldehyde concentrations, crosslink time and immobilization time are 8 mg/mL, 1.0%, 1 h, and 1.5 h, respectively. Moreover, the optimal temperature, pH and thermostability of free and immobilized enzymes were compared. The properties of repeatedly used immobilized aminoacylase were also investigated.
Keywords: Magnetic iron oxide (Fe3O4) nanoparticles; aminoacylase; immobilization; covalent bond
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1. Introduction
Aminoacylase (EC 3.5.1.14), also called N-acyl-L-amino-acid aminohydrolase, is one of the ten most widely used enzymes in the biotechnology field. With strong optic-specific properties, aminoacylase converts L-amino acid acylatehydrolysis to L-amino acid, whereby L-amino acid and D-amino acid acylatehydrolysis can be separated. Aminoacylase-I was first discovered by Schmiedeberg in pig kidneys in 1881 [1]. Although useful, the industrial applications of free aminoacylase must endure its recovery and reuse, along with the difficult product purification process and high cost. Consequently, the immobilization of aminoacylase has been considered an impressive strategy to overcome these obstacles. Numerous studies have investigated immobilized aminoacylase in the separation of alanine, methionine, valine and other amino acids [2-5]. The fact that free aminoacylase can be used for the separation of theanine [6] has garnered interest in determining whether aminoacylase could be immobilized at the nanoscale. Li et al. immobilized aminoacylase on polyvinyl alcohol-based nanofibrous membranes, resulting in high stability and thermostability [7]. However, it is critical to determine whether the immobilized aminoacylase could be employed with magnetic Fe3O4 nanoparticles so that the enzymes could remain quite similar with the corresponding free enzymes in terms of catalytic properties. Magnetic Fe3O4 nanoparticles have been receiving increasing attention in the field of immobilized enzymes due to their numerous advantages, such as simple preparation, strong magnetic response, small size and easy separation from the reaction solution. Magnetic iron oxide nanoparticles have special physical and chemical properties because of their superparamagnetism, quantum size effect and surface-boundary effect. However, the enzymes are difficult to directly immobilize onto magnetic iron oxide nanoparticles. Therefore, several studies have focused on improving the preparation, modification and dispersion of magnetic iron oxide nanoparticles by modifying their surface [8-11]. In 1992, de Cuyper et al. used phospholipid-coated magnetic nanoparticles to immobilize cytochrome-C oxidase, retaining 67% of the activity [12]. In 2008, Yeung et al. developed a new synthesis approach depositing 3 / 21
iron precursor or nanoparticles within the mesopores of MCM-41, which produced encapsulated nanoparticles [13]. However, there is an absence of the immobilization of aminoacylase onto magnetic nanoparticles. By comparison, our study used chemical co-precipitation to synthesize magnetic iron oxide nanoparticles. Ferric chloride and ferrous sulfate were used as the reactants, sodium hydroxide was added drop-wise into the reaction solution, and the magnetic iron oxide nanoparticles were collected by a permanent magnet. We chose APTES [14] to modify the magnetic Fe3O4 nanoparticles because the former can graft with the hydroxyl of the Fe3O4. Then, aminoacylase was immobilized to the Fe3O4-APTES (also called amino-coated Fe3O4) nanoparticles with glutaraldehyde (Schedule 1). Moreover, a protein binding ratio assay and enzyme activity assay were performed on the immobilized aminoacylase.
2. Materials and Methods
2.1 Chemicals Aspergillus oryzae aminoacylase (EC 3.5.1.14) (30,000 U/g) was purchased from Jingchun Biological Technology Co., Ltd. (Shanghai, China). DL-theanine was purchased from Hanhong Chemical Technology Co., Ltd (Shanghai, China). Ferric chloride hexahydrate (FeCl3•6H2O), iron vitriol (FeSO4•7H2O), sodium hydroxide (NaOH), APTES, anhydrous ethanol, ninhydrin, ethylene glycol methyl ether, vitamin C, acetic acid and anhydrous sodium acetate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glutaraldehyde (50%) was purchased from Aibi Chemistry Preparation Co., Ltd. (Shanghai, China).
2.2 Synthesis and surface modification of magnetic Fe3O4 nanoparticles 4 / 21
Magnetic Fe3O4 nanoparticles were prepared by using the conventional co-precipitation method with some modifications [15, 16]. In brief, a mixture of 6.76 g FeCl3•6H2O and 3.48 g FeSO4•7H2O (n(Fe3+):n(Fe2+)=2:1) was dissolved in 100 mL ultrapure water under nitrogen protection. The solution was stirred at 60ºC for 3 h, and its pH was adjusted to 10.0 by the drop-wise addition of 5.0 mol/L NaOH. Subsequently, the magnetite precipitates were separated from the reaction mixture by an external permanent magnet, washed with ultrapure water and ethanol three times, and resuspended in 50% (v/v) ethanol at a final concentration of 10 mg/mL. Because the hydroxyl of the magnetic Fe3O4 nanoparticles is able to react with the oxethyl side chain in APTES, the surface of the magnetic Fe3O4 nanoparticles can be modified by the amino of APTES [14, 17]. Next, 500 µL APTES were added under nitrogen to 150 mL magnetic Fe3O4 nanoparticle solution, which was resuspended above. After stirring at 40ºC for 24 h, the amino-coated Fe3O4 nanoparticles were collected by the permanent magnet, washed three times with ultrapure water and ethanol, and then dried in a vacuum at 60ºC. Finally, the amino-coated Fe3O4 nanoparticles were stored under a seal at 4ºC in dry conditions.
2.3 Immobilization of aminoacylase The amino-coated magnetic Fe3O4 nanoparticles were dissolved into phosphate buffer (0.1 mol/L, pH 7.0) to ensure final concentrations of 4, 6, 8, 10 or 12 mg/mL. Ultrasound was performed on the solution at 40 kHz for 15 min. Then, different concentrations of glutaraldehyde solution, ranging from 0.25% to 2.0% (v/v), were added to the solution, which was then stirred for various lengths of time, ranging from 30 min to 5 h, at 30ºC. The Fe3O4-APTES-glutaraldehyde was collected by the permanent magnet and washed three times with phosphate buffer (0.1 mol/L, pH 7.0) to remove the unreacted glutaraldehyde. The Fe3O4-APTES-glutaraldehyde complexes (final concentration at 8 mg/mL) were then added to the aminoacylase solution (final concentration of 3 mg/mL) for immobilization for various lengths of time, ranging from 15 min to 2 h. Finally, the magnetic Fe3O4-aminoacylase nanoparticles (also called immobilized aminoacylase) were separated by a permanent magnet 5 / 21
and washed three times with phosphate buffer (0.1 mol/L, pH 7.0) to remove free enzymes. As described above, different amounts of Fe3O4-APTES-glutaraldehyde nanoparticles, final glutaraldehyde concentrations, crosslinking times and immobilization times were investigated step by step to reveal the effects of these conditions on the protein binding rate and enzyme activity.
2.4 Characterization of Fe3O4 and Fe3O4-APTES Amino-coated magnetic Fe3O4 nanoparticles were dissolved into different solvents (water, ethanol, methanol, isopropyl alcohol, cyclohexane, toluene, ethyl acetate and petroleum ether) to assess its solubility. For scanning electron microscopy, dried Fe3O4 and Fe3O4-APTES were fixed with KBr on a slide, followed by gold sputtering. The samples were viewed in a HITACHI S4800 electron microscope (Hitachi, Japan) operating at 25 kV. The Fourier transform-infrared spectroscopy (FT-IR) spectrograms were acquired from 16 scans on a Nicolet 6700 infrared spectrometer (Thermo Fisher Scientific, USA) with a test wavelength of 400-4000 cm-1, an interval of 1 cm-1, and excluding background air.
2.5 Protein binding ratio and enzyme activity assay The absorbance was measured by a Libra S22 Spectrophotometer (Biochrom, UK). The absorbance of the aminoacylase solution at 595 nm, both before (ODbefore) and after (ODafter) immobilization, was detected by a spectrophotometer to calculate the protein binding ratio, which is obtained as follows: protein binding ratio = (ODbefore – ODafter)/ODbefore × 100%. Next, 40 mL of phosphate buffer (0.1 mol/L, pH 7.0) were mixed with 5 mL of ultrapure water and 5 mL of 0.2 mol/L DL-theanine in a 250-mL Erlenmeyer flask and kept at 42ºC for 5 min. Then, 20 mg of immobilized aminoacylase or free enzymes were added, and the solution was 6 / 21
vibrated at 42ºC. After 30 min, the reaction was terminated by immediately placing the solution into boiling water. Then, 1 mL the solution was mixed with ninhydrin and fully shaken. The absorbance at 570 nm was obtained to identify the L-theanine content from the standard curve. One unit of aminoacylase activity was defined as the amount of aminoacylase liberated by 1 µmol of L-theanine per hour under the assay conditions (42ºC, pH 7.0). The initial enzyme activity was set as 100%, and the relative enzyme activity was calculated.
2.6 Effects of temperature and pH To obtain the optimal reaction temperature, the relative enzyme activity described in section 2.5 was assessed at different temperatures. An aliquot of 40 mL phosphate buffer (0.1 mol/L, pH 7.0) was mixed with 5 mL of ultrapure water and 5 mL 0.2 mol/L DL-theanine in a 250-EmL Erlenmeyer flask and kept at different temperatures from 29ºC to 72ºC for 5 min. Then, 20 mg of immobilized aminoacylase or free enzyme was added, and the solution was vibrated at 42ºC. After 30 min, the reaction was terminated by immediately placing the solution in boiling water. Then, 1 mL of the solution was mixed with ninhydrin and fully shaken. The absorbance at 570 nm was obtained to identify the L-theanine content from the standard curve. The initial enzyme activity was set as 100%, and the relative enzyme activity was calculated. Moreover, the optimal pH was investigated. The relative enzyme activity assay was performed for immobilized aminoacylase and the free enzyme at its optimal temperature at pH 5.0, 6.0, 7.0, 8.0 and 9.0.
2.7 Stability of the immobilized aminoacylase enzyme The same amount of free or immobilized enzyme was kept at different temperatures (29, 42, 52, 62 and 72ºC) for 1 h; then, the enzyme activity assay described in section 2.5 was performed at the optimal temperature and pH of the given enzyme. The initial enzyme activity (set as 100%) was used to calculate the relative enzyme activity to determine the thermostability. 7 / 21
The activity of immobilized aminoacylase was cyclically measured 6 times. Each time, the immobilized aminoacylase was separated by the permanent magnet and washed three times with phosphate buffer (0.1 mol/L, pH 7.0); the enzyme activity assay was then performed as described in section 2.5. The initial enzyme activity (set as 100%) was used to calculate the relative enzyme activity to determine the stability of the immobilized aminoacylase.
3. Results and discussion
3.1 Synthesis and characterization of Fe3O4 and Fe3O4-APTES The amino-coated Fe3O4 nanoparticles showed high dispersion and stability in hydrophilic solvents, such as water, ethanol, methanol and isopropanol (Figure 1.a left side). However, they were insoluble in hydrophobic solvents, such as cyclohexane, toluene, ethyl acetate and petroleum ether (Figure 1.a right side) due to the hydrophilic amino-coating on the magnetic Fe3O4 nanoparticles [16]. Therefore, the immobilized aminoacylase has potential for application in oil/water reaction systems in industrial production, as the product can be fully collected in the oil phase to guarantee purity, and immobilized aminoacylase can be separated and recycled by the permanent magnet (Figure 1.b). Because of its superparamagnetism, the immobilized aminoacylase, as catalysts, could be applied for capillary microreactors with external magnetic fields [18]. The size and morphology of Fe3O4 and Fe3O4-APTES were observed by SEM (Figures 1.c & 1.d). The unmodified Fe3O4 was composed of clusters of nanoparticles, with sizes ranging from 10 to 30 nm. The size of the Fe3O4-APTES agglomerates ranged from 30 to 70 nm, averaging at 40 nm. Compared with Fe3O4, Fe3O4-APTES exhibits a larger particle diameter and greater uniformity in particle diameter, revealing that the APTES became connected to the magnetic Fe3O4 nanoparticles. 8 / 21
The IR spectra were used to ensure the success of modification (Figure 1.e). From the IR spectra, the absorption peaks at 3420 cm-1 belonged to O-H stretching vibration, whereas those at 1633 cm-1 belonged to O-H deformed vibration. The absorption peak of C-H stretching vibration was located at 2934 cm-1. The peak at 1000 cm-1 belonged to the bending vibration of Si-O bonds. The absorption peak at 584 cm-1 belonged to the stretching vibration mode of Fe-O bonds in Fe3O4. Compared with the IR spectra of Fe3O4 (i), the IR spectra of Fe3O4-APTES (ii) present larger absorption peaks at 584 and 1000 cm-1, which suggests that Fe-O-Si bonds have been formed, whereas the other absorption peaks at 3420, 2934 and 1633 cm-1 show almost no difference [16]. These characteristics indicate the surface modification was successful and that the APTS linked to the surface of the magnetic Fe3O4 nanoparticles.
3.2 Optimal conditions for the immobilization of aminoacylase Different weights of Fe3O4-APTES-glutaraldehyde were used in the immobilization of aminoacylase
to
investigate
the
optimal
Fe3O4-APTES-glutaraldehyde
weight.
The
aminoacylase solution (3 mg/mL) mixed with Fe3O4-APTES-glutaraldehyde was kept at 30ºC for 1.5 h. The protein binding ratio assay and enzyme activity assay were performed (Figure 2.a). As more Fe3O4-APTES-glutaraldehyde was added, the protein binding ratio increased, but the enzyme activity decreased. When Fe3O4-APTES-glutaraldehyde increased to 12 mg/mL, the protein binding ratio reached a maximum of 80%, whereas the relative enzyme activity reduced to 60%. This result is consistent with the results of previous studies [16]. With a limited aminoacylase concentration, more nanoparticles can react with aminoacylase, resulting in a higher protein binding ratio. However, an excessive number of nanoparticles will block the enzyme activity sites, preventing the enzyme activity sites from reacting with the substrates. As a result, the enzyme activity decreased. Therefore, 8 mg/mL was considered the optimal weight of Fe3O4-APTES-glutaraldehyde. Different volumes of 50% (v/v) glutaraldehyde were used in the immobilization of aminoacylase to investigate the optimal glutaraldehyde volume. The protein binding ratio assay 9 / 21
and enzyme activity assay were performed (Figure 2.b). Corresponding to another report [15], when the final glutaraldehyde concentration was less than 1%, the protein binding ratio and enzyme activity increased with increasing glutaraldehyde because glutaraldehyde activated the nanoparticles and increases the protein binding ratio at low concentrations [19]. Moreover, low glutaraldehyde concentrations could improve the uniformity of free enzymes, which allows for a more complete reaction, thereby increasing the relative enzyme activity. The final glutaraldehyde concentration is 1%, and the protein binding ratio and enzyme activity decreased with increasing glutaraldehyde content. With high glutaraldehyde concentrations, the reaction can be complete, only slightly changing the protein binding ratio. However, excessive glutaraldehyde causes a conformational change of aminoacylase, decreasing the relative enzyme activity [20]. Therefore, 1% is considered the final optimal concentration of glutaraldehyde. We measured the protein binding ratio and enzyme activity of different reaction times between Fe3O4-APTES and glutaraldehyde for the immobilization of aminoacylase (Figure 2.c). The protein binding ratio is highest for a 30 min reaction time, coincident with the most activated nanoparticles. As the reaction time increases, the protein binding ratio rapidly decreases because of the instability of glutaraldehyde. Moreover, glutaraldehyde activity declines with increasing time [21]. The enzyme activity reached the maximum level with a reaction time of 1 h, indicating the maximum capacity of aminoacylase. Therefore, a reaction time of 1 h was optimal for Fe3O4-APTES and glutaraldehyde. We examined the protein binding ratio and enzyme activity for different immobilization times between
Fe3O4-APTES-glutaraldehyde
and
aminoacylase
for
the
immobilization
of
aminoacylase (Figure 2.d). The protein binding ratio slightly changed as the immobilization time increased, and it reached a maximum after 1.5 h. Similarly, the enzyme activity reached its maximum after 1.5 h because more nanoparticles and free enzymes connected tightly over time. However, the enzyme activity slightly declined after 1.5 h. When the immobilization time is overly long, many enzyme molecules form a steric hindrance against each other, and the substrate becomes difficult to react with at the enzyme active site, thereby lowering the enzyme activity [22]. Finally, a reaction time of 1.5 h was considered the optimal immobilization time. 10 / 21
3.3 Enzyme activity sensitivity to temperature, pH, thermostability and stability Enzyme activity assays were performed at different temperatures for free and immobilized aminoacylase (Figure 3.a). With the temperature-activity curve, free aminoacylase reached the maximum enzyme activity at 42ºC, whereas immobilized aminoacylase reached the maximum at 52ºC. The immobilized aminoacylase limited the conformational change, causing the enzyme activity site to require more energy to combine with the substrate to initiate the reaction [23]. Moreover, the immobilized enzymes showed their optimimum catalytic activities at higher reaction temperature, which could reduce the mass transfer resistance of the substrate. Enzyme activity assays were performed in solutions with different pH values for free and immobilized aminoacylase (Figure 3.b). Free and immobilized aminoacylase both achieved maximum enzyme activity at pH 7.0. However, both above and below this level, the enzyme activity of immobilized aminoacylase was higher than that of the free enzyme. After immobilization, aminoacylase formed a covalent bond with Fe3O4-APTES-glutaraldehyde, changed the configuration, and increased its relative activity in a broad pH range [24]. Moreover, the nano-particles provided a buffering effect for the enzyme to address the improvement of pH stability [25]. The thermostability of immobilized aminoacylase at different temperatures (29, 42, 52, 62 and 72ºC for 1 h each) was investigated. At 50ºC, both free and immobilized aminoacylase showed slightly reduced activity. However, when the enzymes were kept at 72ºC for 1 h, the activity of free aminoacylase was retained at only 14%, whereas the immobilized aminoacylase remained at approximately 39%. Compared with the free enzyme, the immobilized aminoacylase showed higher thermostability and lower temperature sensitivity [7], which demonstrates its potential for application in the industrial separation of DL-theanine. The number of reuse cycles of immobilized aminoacylase was also investigated (Figure 3.d). After 3 cycles of reuse, the immobilized aminoacylase retained 76% of its activity. After 6 11 / 21
cycles, the immobilized aminoacylase retained 40% of its activity. During recycling, the solution environment disturbed the enzyme conformation, and the nanoparticles slowly lost the aminoacylase molecules, thereby decreasing the enzyme activity. For traditional enzyme immobilization, enzyme molecules are buried in the carrier or bonded covalently to the carrier surface. However, the enzyme activity site can suffer from the blocking effect of the carrier. The space steric hindrance effect further reduces the affinity of enzyme molecules for the substrate. We use Fe3O4 as the carrier core with APTES grafting in our approach, in which the enzyme molecules become stereoscopically cross-linked outside the Fe3O4 nanoparticles. Through the immobilization process, the immobilized enzymes have smaller steric hindrance space and higher affinity for substrate, allowing the immobilized enzyme to be more similar to the free enzyme in terms of catalytic properties. Therefore, the magnetic Fe3O4 nanoparticles used in enzyme immobilization have broad prospects for development and practical application value.
4. Conclusions
In this work, we described a facile method to immobilize aminoacylase onto magnetic Fe3O4 nanoparticles through APTES modification. We optimized the conditions of the immobilization reaction (8 mg/mL Fe3O4, with a 1% final glutaraldehyde concentration after reacting for 1 h and 1.5 h for immobilization), increasing the protein binding rate to 80%, thus increasing the enzyme activity and stability of the immobilized aminoacylase. The immobilized aminoacylase shows favourable thermal and pH stability compared to the free enzyme in the optimum temperature range of 42ºC to 52ºC. In addition, the immobilized aminoacylase displays good reusability (retaining 40% of its activity after 6 recycles) and the convenience of being magnetically recovered. These results confirm that immobilizing enzymes onto iron oxide 12 / 21
nanoparticles by APTES is economical, facile and efficient. Currently, enzyme immobilization is applied in the catalysis field and in other practical fields. The magnetic nanoparticle immobilized enzyme has quite broad research prospects, and we plan to further study related topics.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21476176), the National Key Technology R&D Program of China (No. 2015BAD16B01), High Technology Research and Development Program of China
the National
(863 Program, No.
SS2015AA021002), Science and Technology Commission of Shanghai Municipality (No. 12dz1909403).
Figure Legends:
Schedule 1. Preparation of immobilized aminoacylase on magnetic Fe3O4 nanoparticles. Figure 1. Preparation of immobilized aminoacylase: (a) Solubility; (b) Magnetism; (c) SEM for Fe3O4; (d) SEM for Fe3O4-APTES; (e) IR spectra (blue for Fe3O4 and red for Fe3O4-APTES). Figure 2. Optimization condition for the immobilization of aminoacylase: (a) Different amino-coated Fe3O4 concentrations of 4, 6, 8, 10 or 12 mg/mL, respectively, with 1% (v/v) glutaraldehyde, cross-linked for 1 h and immobilized for 1 h. (b) 2 mg/mL amino-coated Fe3O4 with 0.25-5.0% (v/v) glutaraldehyde, cross-linked for 1 h and immobilized for 1 h. (c) 2 mg/mL 13 / 21
amino-coated Fe3O4 with 1% (v/v) glutaraldehyde, cross-linked for 30 min to 5 h and immobilized for 1 h. (d) 8 mg/mL Fe3O4-APTES-glutaraldehyde for different immobilization times from 15 min to 2 h. Figure 3. Enzyme activity assays with different temperatures, pH, thermostabilities and stability values: (a) Investigation of the optimum temperature of immobilized aminoacylase and the free enzyme in solution at pH=7.0. (b) Investigation of the optimum pH at the optimum temperature. (c) Relative enzyme activity after holding at different temperatures for 1 h. (d) Relative enzyme activity of immobilized aminoacylase after re-use for several cycles.
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The synthesis of Fe3O4 magnetic nanoparticles with APTES modification. Crosslinking of enzyme with less steric effect and more similarity to free enzyme. The optimum temperature of immobilized enzyme was improved. Enhancement of thermal and pH stability, reusability for immobilized aminoacylase.
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S1385-8947(15)01494-1 http://dx.doi.org/10.1016/j.cej.2015.10.083 CEJ 14359
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
14 April 2015 10 October 2015 26 October 2015
Please cite this article as: J. Feng, S. Yu, J. Li, T. Mo, P. Li, Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles, Chemical Engineering Journal (2015), doi: http://dx.doi.org/10.1016/j.cej.2015.10.083
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles
Junchen Feng a, Siran Yu a, Jian Li b, Ting Mo a, Ping Li a,* a
School of Life Sciences and Technology, Tongji University, No. 1239 Siping Road, Shanghai 200092, China
b
School of Engineering, Anhui Agricultural University, No. 130 West Changjiang Road, Hefei 230036, China
* Corresponding author: Prof. Ping Li Tel.: +86 21 65981051 Fax: +86 21 65981041 E-mail address: [email protected]
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Abstract: Aminoacylase (EC 3.5.1.14) was immobilized via covalent bonding to magnetic iron oxide (Fe3O4) nanoparticles with 3-ammonia propyl triethoxy silane (APTES) modification. Magnetic Fe3O4 nanoparticles were prepared by chemical co-precipitation, controlling n(Fe3+):n(Fe2+)=2:1 and c(Fe3+)+c(Fe2+)=0.3 mol/L. The nanoparticles modified with APTES were characterized by SEM and FT-IR. The results showed that the optimal nanoparticle concentration, glutaraldehyde concentrations, crosslink time and immobilization time are 8 mg/mL, 1.0%, 1 h, and 1.5 h, respectively. Moreover, the optimal temperature, pH and thermostability of free and immobilized enzymes were compared. The properties of repeatedly used immobilized aminoacylase were also investigated.
Keywords: Magnetic iron oxide (Fe3O4) nanoparticles; aminoacylase; immobilization; covalent bond
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1. Introduction
Aminoacylase (EC 3.5.1.14), also called N-acyl-L-amino-acid aminohydrolase, is one of the ten most widely used enzymes in the biotechnology field. With strong optic-specific properties, aminoacylase converts L-amino acid acylatehydrolysis to L-amino acid, whereby L-amino acid and D-amino acid acylatehydrolysis can be separated. Aminoacylase-I was first discovered by Schmiedeberg in pig kidneys in 1881 [1]. Although useful, the industrial applications of free aminoacylase must endure its recovery and reuse, along with the difficult product purification process and high cost. Consequently, the immobilization of aminoacylase has been considered an impressive strategy to overcome these obstacles. Numerous studies have investigated immobilized aminoacylase in the separation of alanine, methionine, valine and other amino acids [2-5]. The fact that free aminoacylase can be used for the separation of theanine [6] has garnered interest in determining whether aminoacylase could be immobilized at the nanoscale. Li et al. immobilized aminoacylase on polyvinyl alcohol-based nanofibrous membranes, resulting in high stability and thermostability [7]. However, it is critical to determine whether the immobilized aminoacylase could be employed with magnetic Fe3O4 nanoparticles so that the enzymes could remain quite similar with the corresponding free enzymes in terms of catalytic properties. Magnetic Fe3O4 nanoparticles have been receiving increasing attention in the field of immobilized enzymes due to their numerous advantages, such as simple preparation, strong magnetic response, small size and easy separation from the reaction solution. Magnetic iron oxide nanoparticles have special physical and chemical properties because of their superparamagnetism, quantum size effect and surface-boundary effect. However, the enzymes are difficult to directly immobilize onto magnetic iron oxide nanoparticles. Therefore, several studies have focused on improving the preparation, modification and dispersion of magnetic iron oxide nanoparticles by modifying their surface [8-11]. In 1992, de Cuyper et al. used phospholipid-coated magnetic nanoparticles to immobilize cytochrome-C oxidase, retaining 67% of the activity [12]. In 2008, Yeung et al. developed a new synthesis approach depositing 3 / 21
iron precursor or nanoparticles within the mesopores of MCM-41, which produced encapsulated nanoparticles [13]. However, there is an absence of the immobilization of aminoacylase onto magnetic nanoparticles. By comparison, our study used chemical co-precipitation to synthesize magnetic iron oxide nanoparticles. Ferric chloride and ferrous sulfate were used as the reactants, sodium hydroxide was added drop-wise into the reaction solution, and the magnetic iron oxide nanoparticles were collected by a permanent magnet. We chose APTES [14] to modify the magnetic Fe3O4 nanoparticles because the former can graft with the hydroxyl of the Fe3O4. Then, aminoacylase was immobilized to the Fe3O4-APTES (also called amino-coated Fe3O4) nanoparticles with glutaraldehyde (Schedule 1). Moreover, a protein binding ratio assay and enzyme activity assay were performed on the immobilized aminoacylase.
2. Materials and Methods
2.1 Chemicals Aspergillus oryzae aminoacylase (EC 3.5.1.14) (30,000 U/g) was purchased from Jingchun Biological Technology Co., Ltd. (Shanghai, China). DL-theanine was purchased from Hanhong Chemical Technology Co., Ltd (Shanghai, China). Ferric chloride hexahydrate (FeCl3•6H2O), iron vitriol (FeSO4•7H2O), sodium hydroxide (NaOH), APTES, anhydrous ethanol, ninhydrin, ethylene glycol methyl ether, vitamin C, acetic acid and anhydrous sodium acetate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glutaraldehyde (50%) was purchased from Aibi Chemistry Preparation Co., Ltd. (Shanghai, China).
2.2 Synthesis and surface modification of magnetic Fe3O4 nanoparticles 4 / 21
Magnetic Fe3O4 nanoparticles were prepared by using the conventional co-precipitation method with some modifications [15, 16]. In brief, a mixture of 6.76 g FeCl3•6H2O and 3.48 g FeSO4•7H2O (n(Fe3+):n(Fe2+)=2:1) was dissolved in 100 mL ultrapure water under nitrogen protection. The solution was stirred at 60ºC for 3 h, and its pH was adjusted to 10.0 by the drop-wise addition of 5.0 mol/L NaOH. Subsequently, the magnetite precipitates were separated from the reaction mixture by an external permanent magnet, washed with ultrapure water and ethanol three times, and resuspended in 50% (v/v) ethanol at a final concentration of 10 mg/mL. Because the hydroxyl of the magnetic Fe3O4 nanoparticles is able to react with the oxethyl side chain in APTES, the surface of the magnetic Fe3O4 nanoparticles can be modified by the amino of APTES [14, 17]. Next, 500 µL APTES were added under nitrogen to 150 mL magnetic Fe3O4 nanoparticle solution, which was resuspended above. After stirring at 40ºC for 24 h, the amino-coated Fe3O4 nanoparticles were collected by the permanent magnet, washed three times with ultrapure water and ethanol, and then dried in a vacuum at 60ºC. Finally, the amino-coated Fe3O4 nanoparticles were stored under a seal at 4ºC in dry conditions.
2.3 Immobilization of aminoacylase The amino-coated magnetic Fe3O4 nanoparticles were dissolved into phosphate buffer (0.1 mol/L, pH 7.0) to ensure final concentrations of 4, 6, 8, 10 or 12 mg/mL. Ultrasound was performed on the solution at 40 kHz for 15 min. Then, different concentrations of glutaraldehyde solution, ranging from 0.25% to 2.0% (v/v), were added to the solution, which was then stirred for various lengths of time, ranging from 30 min to 5 h, at 30ºC. The Fe3O4-APTES-glutaraldehyde was collected by the permanent magnet and washed three times with phosphate buffer (0.1 mol/L, pH 7.0) to remove the unreacted glutaraldehyde. The Fe3O4-APTES-glutaraldehyde complexes (final concentration at 8 mg/mL) were then added to the aminoacylase solution (final concentration of 3 mg/mL) for immobilization for various lengths of time, ranging from 15 min to 2 h. Finally, the magnetic Fe3O4-aminoacylase nanoparticles (also called immobilized aminoacylase) were separated by a permanent magnet 5 / 21
and washed three times with phosphate buffer (0.1 mol/L, pH 7.0) to remove free enzymes. As described above, different amounts of Fe3O4-APTES-glutaraldehyde nanoparticles, final glutaraldehyde concentrations, crosslinking times and immobilization times were investigated step by step to reveal the effects of these conditions on the protein binding rate and enzyme activity.
2.4 Characterization of Fe3O4 and Fe3O4-APTES Amino-coated magnetic Fe3O4 nanoparticles were dissolved into different solvents (water, ethanol, methanol, isopropyl alcohol, cyclohexane, toluene, ethyl acetate and petroleum ether) to assess its solubility. For scanning electron microscopy, dried Fe3O4 and Fe3O4-APTES were fixed with KBr on a slide, followed by gold sputtering. The samples were viewed in a HITACHI S4800 electron microscope (Hitachi, Japan) operating at 25 kV. The Fourier transform-infrared spectroscopy (FT-IR) spectrograms were acquired from 16 scans on a Nicolet 6700 infrared spectrometer (Thermo Fisher Scientific, USA) with a test wavelength of 400-4000 cm-1, an interval of 1 cm-1, and excluding background air.
2.5 Protein binding ratio and enzyme activity assay The absorbance was measured by a Libra S22 Spectrophotometer (Biochrom, UK). The absorbance of the aminoacylase solution at 595 nm, both before (ODbefore) and after (ODafter) immobilization, was detected by a spectrophotometer to calculate the protein binding ratio, which is obtained as follows: protein binding ratio = (ODbefore – ODafter)/ODbefore × 100%. Next, 40 mL of phosphate buffer (0.1 mol/L, pH 7.0) were mixed with 5 mL of ultrapure water and 5 mL of 0.2 mol/L DL-theanine in a 250-mL Erlenmeyer flask and kept at 42ºC for 5 min. Then, 20 mg of immobilized aminoacylase or free enzymes were added, and the solution was 6 / 21
vibrated at 42ºC. After 30 min, the reaction was terminated by immediately placing the solution into boiling water. Then, 1 mL the solution was mixed with ninhydrin and fully shaken. The absorbance at 570 nm was obtained to identify the L-theanine content from the standard curve. One unit of aminoacylase activity was defined as the amount of aminoacylase liberated by 1 µmol of L-theanine per hour under the assay conditions (42ºC, pH 7.0). The initial enzyme activity was set as 100%, and the relative enzyme activity was calculated.
2.6 Effects of temperature and pH To obtain the optimal reaction temperature, the relative enzyme activity described in section 2.5 was assessed at different temperatures. An aliquot of 40 mL phosphate buffer (0.1 mol/L, pH 7.0) was mixed with 5 mL of ultrapure water and 5 mL 0.2 mol/L DL-theanine in a 250-EmL Erlenmeyer flask and kept at different temperatures from 29ºC to 72ºC for 5 min. Then, 20 mg of immobilized aminoacylase or free enzyme was added, and the solution was vibrated at 42ºC. After 30 min, the reaction was terminated by immediately placing the solution in boiling water. Then, 1 mL of the solution was mixed with ninhydrin and fully shaken. The absorbance at 570 nm was obtained to identify the L-theanine content from the standard curve. The initial enzyme activity was set as 100%, and the relative enzyme activity was calculated. Moreover, the optimal pH was investigated. The relative enzyme activity assay was performed for immobilized aminoacylase and the free enzyme at its optimal temperature at pH 5.0, 6.0, 7.0, 8.0 and 9.0.
2.7 Stability of the immobilized aminoacylase enzyme The same amount of free or immobilized enzyme was kept at different temperatures (29, 42, 52, 62 and 72ºC) for 1 h; then, the enzyme activity assay described in section 2.5 was performed at the optimal temperature and pH of the given enzyme. The initial enzyme activity (set as 100%) was used to calculate the relative enzyme activity to determine the thermostability. 7 / 21
The activity of immobilized aminoacylase was cyclically measured 6 times. Each time, the immobilized aminoacylase was separated by the permanent magnet and washed three times with phosphate buffer (0.1 mol/L, pH 7.0); the enzyme activity assay was then performed as described in section 2.5. The initial enzyme activity (set as 100%) was used to calculate the relative enzyme activity to determine the stability of the immobilized aminoacylase.
3. Results and discussion
3.1 Synthesis and characterization of Fe3O4 and Fe3O4-APTES The amino-coated Fe3O4 nanoparticles showed high dispersion and stability in hydrophilic solvents, such as water, ethanol, methanol and isopropanol (Figure 1.a left side). However, they were insoluble in hydrophobic solvents, such as cyclohexane, toluene, ethyl acetate and petroleum ether (Figure 1.a right side) due to the hydrophilic amino-coating on the magnetic Fe3O4 nanoparticles [16]. Therefore, the immobilized aminoacylase has potential for application in oil/water reaction systems in industrial production, as the product can be fully collected in the oil phase to guarantee purity, and immobilized aminoacylase can be separated and recycled by the permanent magnet (Figure 1.b). Because of its superparamagnetism, the immobilized aminoacylase, as catalysts, could be applied for capillary microreactors with external magnetic fields [18]. The size and morphology of Fe3O4 and Fe3O4-APTES were observed by SEM (Figures 1.c & 1.d). The unmodified Fe3O4 was composed of clusters of nanoparticles, with sizes ranging from 10 to 30 nm. The size of the Fe3O4-APTES agglomerates ranged from 30 to 70 nm, averaging at 40 nm. Compared with Fe3O4, Fe3O4-APTES exhibits a larger particle diameter and greater uniformity in particle diameter, revealing that the APTES became connected to the magnetic Fe3O4 nanoparticles. 8 / 21
The IR spectra were used to ensure the success of modification (Figure 1.e). From the IR spectra, the absorption peaks at 3420 cm-1 belonged to O-H stretching vibration, whereas those at 1633 cm-1 belonged to O-H deformed vibration. The absorption peak of C-H stretching vibration was located at 2934 cm-1. The peak at 1000 cm-1 belonged to the bending vibration of Si-O bonds. The absorption peak at 584 cm-1 belonged to the stretching vibration mode of Fe-O bonds in Fe3O4. Compared with the IR spectra of Fe3O4 (i), the IR spectra of Fe3O4-APTES (ii) present larger absorption peaks at 584 and 1000 cm-1, which suggests that Fe-O-Si bonds have been formed, whereas the other absorption peaks at 3420, 2934 and 1633 cm-1 show almost no difference [16]. These characteristics indicate the surface modification was successful and that the APTS linked to the surface of the magnetic Fe3O4 nanoparticles.
3.2 Optimal conditions for the immobilization of aminoacylase Different weights of Fe3O4-APTES-glutaraldehyde were used in the immobilization of aminoacylase
to
investigate
the
optimal
Fe3O4-APTES-glutaraldehyde
weight.
The
aminoacylase solution (3 mg/mL) mixed with Fe3O4-APTES-glutaraldehyde was kept at 30ºC for 1.5 h. The protein binding ratio assay and enzyme activity assay were performed (Figure 2.a). As more Fe3O4-APTES-glutaraldehyde was added, the protein binding ratio increased, but the enzyme activity decreased. When Fe3O4-APTES-glutaraldehyde increased to 12 mg/mL, the protein binding ratio reached a maximum of 80%, whereas the relative enzyme activity reduced to 60%. This result is consistent with the results of previous studies [16]. With a limited aminoacylase concentration, more nanoparticles can react with aminoacylase, resulting in a higher protein binding ratio. However, an excessive number of nanoparticles will block the enzyme activity sites, preventing the enzyme activity sites from reacting with the substrates. As a result, the enzyme activity decreased. Therefore, 8 mg/mL was considered the optimal weight of Fe3O4-APTES-glutaraldehyde. Different volumes of 50% (v/v) glutaraldehyde were used in the immobilization of aminoacylase to investigate the optimal glutaraldehyde volume. The protein binding ratio assay 9 / 21
and enzyme activity assay were performed (Figure 2.b). Corresponding to another report [15], when the final glutaraldehyde concentration was less than 1%, the protein binding ratio and enzyme activity increased with increasing glutaraldehyde because glutaraldehyde activated the nanoparticles and increases the protein binding ratio at low concentrations [19]. Moreover, low glutaraldehyde concentrations could improve the uniformity of free enzymes, which allows for a more complete reaction, thereby increasing the relative enzyme activity. The final glutaraldehyde concentration is 1%, and the protein binding ratio and enzyme activity decreased with increasing glutaraldehyde content. With high glutaraldehyde concentrations, the reaction can be complete, only slightly changing the protein binding ratio. However, excessive glutaraldehyde causes a conformational change of aminoacylase, decreasing the relative enzyme activity [20]. Therefore, 1% is considered the final optimal concentration of glutaraldehyde. We measured the protein binding ratio and enzyme activity of different reaction times between Fe3O4-APTES and glutaraldehyde for the immobilization of aminoacylase (Figure 2.c). The protein binding ratio is highest for a 30 min reaction time, coincident with the most activated nanoparticles. As the reaction time increases, the protein binding ratio rapidly decreases because of the instability of glutaraldehyde. Moreover, glutaraldehyde activity declines with increasing time [21]. The enzyme activity reached the maximum level with a reaction time of 1 h, indicating the maximum capacity of aminoacylase. Therefore, a reaction time of 1 h was optimal for Fe3O4-APTES and glutaraldehyde. We examined the protein binding ratio and enzyme activity for different immobilization times between
Fe3O4-APTES-glutaraldehyde
and
aminoacylase
for
the
immobilization
of
aminoacylase (Figure 2.d). The protein binding ratio slightly changed as the immobilization time increased, and it reached a maximum after 1.5 h. Similarly, the enzyme activity reached its maximum after 1.5 h because more nanoparticles and free enzymes connected tightly over time. However, the enzyme activity slightly declined after 1.5 h. When the immobilization time is overly long, many enzyme molecules form a steric hindrance against each other, and the substrate becomes difficult to react with at the enzyme active site, thereby lowering the enzyme activity [22]. Finally, a reaction time of 1.5 h was considered the optimal immobilization time. 10 / 21
3.3 Enzyme activity sensitivity to temperature, pH, thermostability and stability Enzyme activity assays were performed at different temperatures for free and immobilized aminoacylase (Figure 3.a). With the temperature-activity curve, free aminoacylase reached the maximum enzyme activity at 42ºC, whereas immobilized aminoacylase reached the maximum at 52ºC. The immobilized aminoacylase limited the conformational change, causing the enzyme activity site to require more energy to combine with the substrate to initiate the reaction [23]. Moreover, the immobilized enzymes showed their optimimum catalytic activities at higher reaction temperature, which could reduce the mass transfer resistance of the substrate. Enzyme activity assays were performed in solutions with different pH values for free and immobilized aminoacylase (Figure 3.b). Free and immobilized aminoacylase both achieved maximum enzyme activity at pH 7.0. However, both above and below this level, the enzyme activity of immobilized aminoacylase was higher than that of the free enzyme. After immobilization, aminoacylase formed a covalent bond with Fe3O4-APTES-glutaraldehyde, changed the configuration, and increased its relative activity in a broad pH range [24]. Moreover, the nano-particles provided a buffering effect for the enzyme to address the improvement of pH stability [25]. The thermostability of immobilized aminoacylase at different temperatures (29, 42, 52, 62 and 72ºC for 1 h each) was investigated. At 50ºC, both free and immobilized aminoacylase showed slightly reduced activity. However, when the enzymes were kept at 72ºC for 1 h, the activity of free aminoacylase was retained at only 14%, whereas the immobilized aminoacylase remained at approximately 39%. Compared with the free enzyme, the immobilized aminoacylase showed higher thermostability and lower temperature sensitivity [7], which demonstrates its potential for application in the industrial separation of DL-theanine. The number of reuse cycles of immobilized aminoacylase was also investigated (Figure 3.d). After 3 cycles of reuse, the immobilized aminoacylase retained 76% of its activity. After 6 11 / 21
cycles, the immobilized aminoacylase retained 40% of its activity. During recycling, the solution environment disturbed the enzyme conformation, and the nanoparticles slowly lost the aminoacylase molecules, thereby decreasing the enzyme activity. For traditional enzyme immobilization, enzyme molecules are buried in the carrier or bonded covalently to the carrier surface. However, the enzyme activity site can suffer from the blocking effect of the carrier. The space steric hindrance effect further reduces the affinity of enzyme molecules for the substrate. We use Fe3O4 as the carrier core with APTES grafting in our approach, in which the enzyme molecules become stereoscopically cross-linked outside the Fe3O4 nanoparticles. Through the immobilization process, the immobilized enzymes have smaller steric hindrance space and higher affinity for substrate, allowing the immobilized enzyme to be more similar to the free enzyme in terms of catalytic properties. Therefore, the magnetic Fe3O4 nanoparticles used in enzyme immobilization have broad prospects for development and practical application value.
4. Conclusions
In this work, we described a facile method to immobilize aminoacylase onto magnetic Fe3O4 nanoparticles through APTES modification. We optimized the conditions of the immobilization reaction (8 mg/mL Fe3O4, with a 1% final glutaraldehyde concentration after reacting for 1 h and 1.5 h for immobilization), increasing the protein binding rate to 80%, thus increasing the enzyme activity and stability of the immobilized aminoacylase. The immobilized aminoacylase shows favourable thermal and pH stability compared to the free enzyme in the optimum temperature range of 42ºC to 52ºC. In addition, the immobilized aminoacylase displays good reusability (retaining 40% of its activity after 6 recycles) and the convenience of being magnetically recovered. These results confirm that immobilizing enzymes onto iron oxide 12 / 21
nanoparticles by APTES is economical, facile and efficient. Currently, enzyme immobilization is applied in the catalysis field and in other practical fields. The magnetic nanoparticle immobilized enzyme has quite broad research prospects, and we plan to further study related topics.
Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21476176), the National Key Technology R&D Program of China (No. 2015BAD16B01), High Technology Research and Development Program of China
the National
(863 Program, No.
SS2015AA021002), Science and Technology Commission of Shanghai Municipality (No. 12dz1909403).
Figure Legends:
Schedule 1. Preparation of immobilized aminoacylase on magnetic Fe3O4 nanoparticles. Figure 1. Preparation of immobilized aminoacylase: (a) Solubility; (b) Magnetism; (c) SEM for Fe3O4; (d) SEM for Fe3O4-APTES; (e) IR spectra (blue for Fe3O4 and red for Fe3O4-APTES). Figure 2. Optimization condition for the immobilization of aminoacylase: (a) Different amino-coated Fe3O4 concentrations of 4, 6, 8, 10 or 12 mg/mL, respectively, with 1% (v/v) glutaraldehyde, cross-linked for 1 h and immobilized for 1 h. (b) 2 mg/mL amino-coated Fe3O4 with 0.25-5.0% (v/v) glutaraldehyde, cross-linked for 1 h and immobilized for 1 h. (c) 2 mg/mL 13 / 21
amino-coated Fe3O4 with 1% (v/v) glutaraldehyde, cross-linked for 30 min to 5 h and immobilized for 1 h. (d) 8 mg/mL Fe3O4-APTES-glutaraldehyde for different immobilization times from 15 min to 2 h. Figure 3. Enzyme activity assays with different temperatures, pH, thermostabilities and stability values: (a) Investigation of the optimum temperature of immobilized aminoacylase and the free enzyme in solution at pH=7.0. (b) Investigation of the optimum pH at the optimum temperature. (c) Relative enzyme activity after holding at different temperatures for 1 h. (d) Relative enzyme activity of immobilized aminoacylase after re-use for several cycles.
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The synthesis of Fe3O4 magnetic nanoparticles with APTES modification. Crosslinking of enzyme with less steric effect and more similarity to free enzyme. The optimum temperature of immobilized enzyme was improved. Enhancement of thermal and pH stability, reusability for immobilized aminoacylase.
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