Abstract
Magnetic iron dust, a byproduct by many chemical industries that performs the reduction of nitro compounds to amine, was used for laccase immobilization. The characterization of magnetic iron dust was done by X-ray diffraction, Fourier-transform infrared, and dynamic light scattering. Biodegradable polymer, chitosan, was coated on to the magnetic iron dust by reverse phase suspension method, which was confirmed by Fourier-transform infrared analysis. Immobilization of the laccase enzyme was done onto the chitosan-coated and non-coated magnetic iron dust. The immobilization was monitored by Fourier-transform infrared analysis. Binding efficiency, optimum pH, and optimum temperature for these immobilized laccases were investigated. X-ray diffraction pattern of magnetic iron dust confirmed presence of magnetite (Fe3O4) and maghemite (γ-Fe2O3) with a particle size of 529.6 nm measured by dynamic light scattering. Laccase was immobilized on chitosan-coated and non-coated magnetic iron dust, monitored by Fourier-transform infrared spectra. Binding efficiency of the laccases was found to be 100% onto the coated and non-coated magnetic iron dust and their activity remained to be 63% and 82%, respectively, even after the 10th cycle of their use. The present results demonstrated the applicability of these immobilized laccase system in the industry in terms of their reusability and waste recycling.
Introduction
In recent years, nano-sized magnetic particles, among them, maghemite and magnetite have been studied for their applicability in the area of biological sciences. Some of the recent applications of these particles include target-based drug delivery, DNA–RNA isolation and purification, and protein immobilization on solid phase support, among others (Sun et al., 2005). Enzymes that are immobilized on solid phase support offer various advantages in terms of their recyclability and recovery than their soluble counterparts (Sheldon, 2007). Immobilized enzymes on magnetic nano-particles provide an advantage in that there is less recovery time in magnetic environment and they offer less mechanical damage to the enzyme (Jiang et al., 2009). Other important advantages of immobilization include high surface area for binding higher amount of enzyme (Dyal et al., 2003), lower mass transfer resistance (Lee et al., 2005), lower operational cost (Lan et al., 2008), less diffusion (Rossi et al., 2004), and low cost (Sahoo et al., 2011).
The presence of functional groups on nano particle surfaces is crucial for immobilizing bio-active reagents. The loading capacity and stability of the bioactive molecule depends on the nature of these functional groups’ presence on the surface of magnetic particles (Jia et al., 2003). Synthetic and natural polymers are employed for increasing the functionality of magnetic particles (Lu et al., 2007). Chitosan is an example of such a polymer that is widely used for similar applications (Li et al., 2008). Recently, many research groups have developed chitosan-based immobilization systems for enzymes (Agnihotri et al., 2004).
Laccases (benzenediol: oxidoreductase, EC 1.10.3.2) are extracellular enzymes found in plants, insects, and many microorganisms (mostly fungi; Rodríguez Couto and Toca Herrera, 2006). These classes of enzymes use radical catalyzed reactions to oxidize aromatic and non-aromatic compounds using molecular oxygen (Wong and Yu, 1999). The high reaction potential and broad substrate specificity of these enzymes make them suitable for industrial applications such as textile dye bleaching, pulp bleaching, food improvement, bioremediation (Riva, 2006), among others. The laccases have been immobilized using non-magnetic micro scale chitosan-coated materials to enhance their reusability and efficiency. Many of them are used in various industrial applications (Kalkan et al., 2012). The recovery of such an immobilized enzyme system is often achieved using filtration and centrifugation. This process induces mechanical stress on the enzyme and results in decrease or loss of activity. To overcome this hurdle, magnetic nano-particle based supports are currently in use for easy recovery of the immobilized enzymes from the applied media with minimum loss of the activity (Ansari and Husain, 2012).
The hypothesis of the present study is that laccase enzyme, which has multiple industrial applications, would have higher efficiency and better catalytic activity upon binding with magnetic iron dust (MID), an industrial waste. The objectives of the study were to characterize the MID by modern tools like X-ray diffraction (XRD), Fourier-transform infrared (FT-IR), and dynamic light scattering (DLS), surface modification of MID by chitosan coating and subsequent immobilization of the laccase enzyme, and last, measurement of the kinetic parameters like optimum pH and optimum temperature range for the immobilized enzyme.
Materials and methods
Materials
MID that was provided by Asha Nitrochem Industries Ltd, Dadar East, Mumbai-400014, India. Laccase (E.C.1.10.3.2) from Trametes versicolor. ABTS [2,2-azinobis (3-ethylbenzenethiazoline-6-sulphonic-acid)], carbodiimide, EDAC, (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride), glutaraldehyde, and chitosan powder were purchased from Sigma-Aldrich. Glacial acetic acid, paraffin oil (light), tween 80, sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased from SRL, India. Folin-Ciocalteu’s reagent was purchased from Merck India, Ltd. Bovine serum albumin was purchased from HI-MEDIA, India. All reagents used in this study were of analytical grade.
Instrumental analysis
XRD (D2-Phaser 205072, Bruker, Germany) was used to analyze the type of crystal structure of the MID and identify the sample. Particle size analysis of MID was done using DLS (Zetasizer Nano ZS, Malvern, UK). The properties of chitosan-coated and uncoated MID was evaluated by FT-IR spectra recorded on a Nicolet 60 SXB spectrophotometer (Nicolet, USA).
Characterization of MID
MID was characterized using D2-phaser powder X-ray diffractometer. Results showed diffracted lines that correspond to different plane or miller indices of the crystal. Data were recorded in the 2θ range of 10℃ to 70℃ in steps of 0.02°. Thus, in Bragg’s law, the angles θ that were tested are 5 to 40°. The 2θ angles were sampled in increasing steps of 0.02°, counting at each angle for 1.2 s.
The size of the MID was determined by a DLS Particle Size Analyzer from 25℃ to 45℃. The measuring range was from 1 nm to 6 µm and the light source was a 633 nm He-Ne laser of 4 mW. MID were dispersed in water by mild sonication using a bath sonicator. The particle size of the dispersed MID was then measured.
Preparation of chitosan-coated MID
Coating of MID with chitosan was done using reverse phase suspension technique by modifying the procedure as described by Kalkan et al. (2012). In this procedure, 1000 mg MID was washed three times with 1% HCl by mild sonication using a bath sonicator and thrice with ethanol to remove the impurities. The washed MID was then dried in an oven at 50℃.
For the chitosan coating, 700.0 mg of ethanol-washed MID was added to 150.0 ml paraffin oil containing 1.5 ml tween 80 in a round-bottomed flask. Tween 80 was added to the MID drop wise while continuously stirring with the glass rod. To this mixture, 9.0 ml of glutaraldehyde (25% in dH2O) was added and kept on an orbital shaker at 100 rpm for 24 hours. The resultant chitosan-coated MID was separated from the reaction mixture by a permanent magnet, washed several times with acetone, and dried in vacuum oven at 40℃.
Characterization of chitosan-coated and uncoated MID by FT-IR
The functional groups of uncoated MID and chitosan-coated MID were detected by FT-IR analysis. The samples were prepared in KBr disc and then the FT-IR spectra were recorded by an FT-IR spectrometer (Thermo Scientific NICOLET 6700, USA) between 4000 and 400 cm−1 wavenumber.
Activation of uncoated/chitosan-coated MID with EDAC (carbodiimide)
The activation of chitosan-coated MID with EDAC was done by using a modified method of Kalkan et al. (2012). For activation with EDAC, 100.0 mg of chitosan-coated MID were washed twice with 2.0 ml of phosphate buffer (0.03 M; pH 6.0). Hundred milligrams of washed chitosan-coated MID dispersed in 2.0 ml phosphate buffer (0.03 M, pH 6.0), and to this 0.5 ml EDAC solution (2.5% w/v in same phosphate buffer) was added. The mixture was then sonicated by an ultrasonic bath for 30 min at room temperature. The mixture was stirred by a magnetic stirrer at 1000 rpm, at 4℃ for 6 hours and stored at the same temperature overnight. The EDAC activated chitosan-coated MID was then incubated at 4℃ for 24 hours. Prior to its use, the EDAC activated chitosan-coated MID was recovered by placing the container on a strong permanent magnet and the supernatant was removed.
Laccase immobilization onto chitosan-coated and uncoated MID
Laccase was immobilized onto EDAC-activated chitosan-coated and uncoated MID. For this specific purpose, 100.0 mg of each type of MID was washed using 2.0 ml phosphate buffer (PB, pH 6.0). Two milliliters of laccase solution (0.1–0.8 mg/ml in PB, pH 6.0) was added to each type of washed MID. The system was sonicated for 3 minutes. The reaction mixture was stored at 4℃ and sonicated two more times at one hour intervals to ensure uniform dispersion and then stored at 4℃ overnight. Laccase immobilized systems, laccase bound chitosan-coated and uncoated MID, were then separated from the solution by a strong magnet and each system was washed with PB until the unbound enzyme was completely removed. The presence of laccase on MID was detected by FT-IR spectra.
Immobilization efficiency
Immobilization efficiency of laccase on to EDAC activated chitosan-coated and uncoated MID was estimated using the modified Folin-Lowry assay. Bovine serum albumin (BSA) was used as the standard protein. The modified Folin-Lowry assay is a spectroscopic analytical method routinely used to measure the concentration of a protein in a given solution (Sung and Bae, 2000). To determine the immobilized amount of the laccase, the concentrations of the enzyme in the initial solution and in the washing solutions after the immobilization process were detected by modified Folin-Lowry assay. The enzyme concentrations in the solutions were determined from the BSA calibration curve. The amount of the immobilized enzymes was calculated from the differences of the amounts of initial enzyme and the unbound enzyme detected in the washing solutions.
Measurement of laccase activity
The activity of laccase was assayed by double beam UV–Vis spectrophotometer-2203, Systronics (India) Limited. Free and immobilized laccase activities were determined against ABTS and formation of green ABTS radical cation (ABTS+) upon oxidation of ABTS by laccase (Hublik and Schinner, 2000).
For the measurement of free laccase activity, 20 µl laccase solution (1 mg/ml, citrate buffer, pH 3.0) was added into 2.98 ml citrate buffer and the enzymatic reaction was initiated by adding 20 µl ABTS solution (0.25 mM into the citrate buffer; pH 3.0) into the assay medium at 25℃ temperature and the activity was noted after every 10 seconds at 420 nm. Laccase activity was calculated from the initial slope of the curve obtained between 10 and 60 seconds of reaction. One unit activity of laccase is defined as the amount of laccase required to oxidize 1 µmol of ABTS per minute under the described conditions.
For the determination of immobilized laccase activity, 100.0 mg immobilized laccase containing MID particles were taken and added in 3.0 ml citrate buffer (pH 3.0). The enzymatic reaction was initiated by adding 20 µl ABTS solution (0.25 mM into the citrate buffer; pH 3.0) into the assay medium at room temperature and the activity was noted after every 10 seconds at 420 nm. The assay was accomplished in cuvette (3.5 ml capacity) and then a strong magnet was fitted in spectrophotometer near the cuvette so that during the assay, the MID would get attached to the walls of the cuvette and hence not interfere with the assay.
Effect of pH
The effect of pH on the activities of free and immobilized laccase was determined by measuring the laccase activity against 0.25 mM ABTS. A pH range of 2.6 to 7.0 was chosen to obtain an optimum pH. Citrate phosphate buffer (0.1 M) at pH 2.6, 6.6, and 7.0, and citrate buffer (0.1 M) in a pH range of 3.0–6.2, at 25℃ were used.
Effect of temperature
The effect of temperature on the free and immobilized laccase activity was determined by assaying the laccase activity at different temperatures in the range of 20–80℃ against 0.25 mM ABTS at pH 5.0.
Reusability stability and storage stability
The reusability stability of immobilized laccase was studied by detecting the residual activity. At the end of each cycle, the immobilized laccase was washed thrice with citrate buffer to remove any residual substrate on the immobilized laccase surface. They were then reintroduced into a fresh reaction medium and laccase activity was measured. The storage stability was determined by storing the free and immobilized enzymes at 4℃ temperature for 1 month with laccase activity being measured every day.
Results and discussion
Characterization of MID by XRD and DLS
MID was provided by Asha Nitrochem Industries Ltd and characterized using D2-phaser powder X-ray diffractometer. During the investigation done by L. Wang et al., they found that a variety of nitro-arenes were reduced to the corresponding anilines by nanosized activated metallic iron powder in water at 210℃. During this conversion, hydrogen gas was liberated and a brown precipitate of Fe3O4 was formed from activated metallic iron powder (Wang et al., 2009).
The lines seen in the XRD pattern of the MID sample correspond to crystalline phases. The line pattern confirmed the presence of mixed phases in the MID (Figure 1). The detailed analysis has been carried out based on Crystallography Open Database (COD). All the crystallite lines identified with respective phase(s) are shown in Figure 1. Details of compositions along with its peak positions are summarized in Table 1. Analysis of the pattern showed the dominance of magnetite (Fe3O4) and maghemite (γ-Fe2O3) phases in the sample. Partial presence of goethite phase was also observed. Intensity distribution showed that MID was mostly composed of magnetite phase along with hematite (α-Fe2O3) as secondary phase and goethite as impurity phase. Particle size analysis of MID was done using DLS. The particle size of the dispersed MID were measured to be 529.6 nm.
XRD pattern of MID sample. Details of MID compositions along with its peak positions.
Characterization of chitosan-coated and uncoated MID by FT-IR
The coating of chitosan on MID was confirmed by FT-IR spectroscopic analysis. As can be seen in the FT-IR spectrum of MID coated with chitosan (Figure 2(b)), a sharp peak at 1630 cm−1 was observed, which was attributed to the free –NH2 group of chitosan; a peak due to –OH group of chitosan polymer at ∼3425 cm−1 was present in the case of the magnetic chitosan. Presence of peaks at 2923 cm−1 and ∼2875 cm−1, corresponding to C–H stretching absorption was due to cross-linked chitosan. Similarly, two peaks at ∼2960 cm−1 and ∼2870 cm−1, corresponding to C–H stretching absorptions were also present, which resulted to the cross-linked chitosan. The spectrum of uncoated MID (Figure 2(a)) and chitosan-coated MID (Figure 2(b)) showed peaks at ∼562 and ∼635 cm−1, indicating the presence of magnetic component (Fe–O). Similarly, Häfeli et al. reported two peaks at ∼577 and ∼637 cm−1 due to stretching vibration of Fe–O at the tetrahedral position (Häfeli, 2004).
FT-IR spectra of MID (a) and chitosan-coated MID (b).
Optimization of laccase immobilization on chitosan-coated and uncoated MID and binding efficiency
The efficiency of an enzyme can be enhanced by immobilizing it on solid phase support. In the present study, the laccases, having many industrial applications, were immobilized onto magnetically separable MID. The immobilization onto chitosan-coated and uncoated MID were done after the activation of chitosan-coated and uncoated MID with EDAC.
Activity values for varying ratios of bound laccase enzyme to uncoated MID.
Note: Bold values indicate the best results.
Activity values for varying ratios of bound laccase enzyme to chitosan-coated MID.
Note: Bold values indicate the best results.
Different weight ratios of laccase chitosan-coated and uncoated MID, ranging from 0.002 to 0.016, were taken in the study (see Tables 2 and 3). It was observed that the activity of all varying ratios of laccase immobilized onto chitosan-coated MID was higher compared with activity of laccase immobilized onto uncoated MID. Maximum activity (1.44 U/mg) of laccase immobilized on uncoated MID was obtained at 0.008 weight ratio (Table 2). Similarly, the maximum activity (1.85 U/mg) of laccase immobilized on chitosan-coated MID was also obtained at 0.008 weight ratio (Table 3).
Binding confirmation
The binding of the laccase enzyme to chitosan-coated and uncoated MID was confirmed by FT-IR spectroscopy. Figure 3 shows the FT-IR spectra for free laccase, laccase bound uncoated MID, and laccase bound chitosan-coated MID.
FT-IR spectra of free laccase (a), laccase bound uncoated MID (b), and laccase bound chitosan-coated MID (c).
As seen in the FT-IR spectrum, the peak between 3700 and 3000 cm−1 corresponds to stretching vibrations of O–H and N–H. The spectrum of laccase bound uncoated MID and laccase bound chitosan-coated MID, has peaks ∼566 and ∼1622 cm−1, indicating the presence of magnetic component (Fe–O) and the –C = N– vibration resulting from the reaction between amine groups of laccase, chitosan, and aldehyde group of glutaraldehyde during crosslinking. The peaks at 2922 cm−1 and 2853 cm−1 confirmed the presence of chitosan as well as laccase in the spectra of laccase bound chitosan-coated MID (Figure 3(c)).
Effect of pH on enzyme activity
The effect of pH on the activity of the free and immobilized laccase were examined within a pH range of 2.6–7.0 at 30℃ and the obtained relative activities were presented in Figure 4. The immobilized enzyme showed an increasing activity at the higher pH compared with the free enzyme. The optimum pH for free laccase (L), immobilized laccase on uncoated MID, and chitosan-coated MID were found to be 3.0, 4.6, and 5.4, respectively. Immobilization resulted in 1.6 or 2.4 unit shift to higher pH values for the systems. The analogous results have been observed by other investigator (Hu et al., 2009). This was mostly due to the variations in the conformation of the enzyme upon adsorption or covalent bonds formation and change in microenvironment upon immobilization. A shift in the optimum pH toward higher values was reported for the lipase immobilized onto chitosan microspheres activated by carbodiimide (Zhang et al., 2013) and invertase immobilized onto poly (2-hydroxyethyl methacrylate) activated by CC (Arica and Hasirci, 1987).
Effect of pH on the enzyme activities of free laccase, laccase immobilized on uncoated MID, and laccase immobilized on chitosan-coated MID.
It was demonstrated that all immobilized laccase retained more than 70% of their original activities between pH 4.2 and 5.4 while for free laccase, activity was decreased to 45% at pH 4.2. A high maximum activity of the immobilized enzyme systems was expected, because immobilization could make the enzyme more stable against pH changes. Results showed that immobilized laccase could be used more efficiently and repeatedly in the defined pH region compared with the free laccase.
Effect of temperature
The effect of temperature on the activities of free and immobilized laccase were examined in the range of 20–80℃. The maximum activities of free and immobilized laccase against temperature are represented in Figure 5.
Effect of temperature on the enzyme activities of free laccase, laccase immobilized on uncoated MID, and laccase immobilized on chitosan-coated MID.
The optimum temperature for free laccase (L), the immobilized laccases uncoated MID, and the immobilized laccases chitosan-coated MID were found to be 40℃, 50℃, and 60℃, respectively. Such changes in the optimum temperature values of the immobilized enzymes compared with the free enzymes depend on the immobilization method and the interactions between the enzyme and the support. Early reports claimed shifts in the optimum temperature of the bound immobilized laccases. It was observed that free laccases exhibited more than 80% of their original activities between temperature range of 30–60℃, immobilized laccases on uncoated MID demonstrated more than 80% of their original activities between temperature range of 40–70℃ and immobilized laccase on chitosan-coated MID demonstrated more than 80% of their original activities between temperature range of 30–70℃.
Reusability of immobilized laccase
Reusability of immobilized enzymes is the most important aspect for industrial applications. The underlying reason is that immobilized enzymes decrease the cost of production because of their repeated, continuous, and batch use (Stloukal et al., 2014). The activity of immobilized laccase was evaluated using a repeated batch process (10 times in a day) to observe the reuse of the immobilized enzyme (Figure 6). After the fifth cycle, the immobilized laccase on uncoated MID and immobilized laccase on chitosan-coated MID showed 92% and 86% activity, respectively. At the 10th use, the retained activities for immobilized laccase on uncoated MID and immobilized laccase on chitosan-coated MID systems were found to be more than 60% for both immobilized systems; these values were 63% and 82%, respectively. The decrease in activity might be because of leakage of immobilized enzyme with the process and washing steps at the end of each cycle. The higher activities were obtained for covalently immobilized systems (immobilized laccase on chitosan-coated MID) due to the strong chemical interactions between the enzyme and the support. On the other hand, comparatively less activities were found for laccase immobilized on uncoated MID by adsorption. This might be due to leakage of adsorbed enzyme during the process and washing step. In some recent reports, the activity of the enzyme was retained at 80% after 10 batch uses for the laccase immobilized by adsorption and crosslinking onto magnetic microspheres (Hu et al., 2014) and 70% after 10 consecutive operations for laccase immobilized on magnetic mesoporous silica spheres by covalent binding method (Zhu et al., 2007).
Effect of reuse number on the enzyme activities of laccase immobilized on uncoated MID and laccase immobilized on chitosan-coated MID.
Storage stability of immobilized laccase
Comparison of storage stability of free and immobilized laccase.
Conclusion
The present study demonstrated the use of MID, a byproduct from chemical industries, as a solid support for immobilizing enzymes for industrial applications. The results presented here clearly show that the MID, which was found to contain magnetite and maghemite as the major constituents, can be successfully used to immobilize laccases on its coated and non-coated surface. The use of MID enhanced the working pH and temperature ranges for the enzyme by providing stability to the enzyme via strong chemical bonding. MID increased the scope of reusability of the immobilized enzyme and thereby increasing its potential as support for immobilization.
Footnotes
Acknowledgements
The authors would like to extend their appreciation to the Asha Nitrochem Industries Ltd for providing magnetic iron dust.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by CHARUSAT University.